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Heparin causes platelet dysfunction and induces fibrinolysis before cardiopulmonary bypass
Heparin Causes Platelet Dysfunction and Induces Fibrinolysis Before Cardiopulmonary Bypass Shukri F. Khuri, MD, C. Robert Valeri, MD, Joseph Loscalzo, MD, PhD, Mark J. Weinstein, PhD, Vladimir Birjiniuk, MD, Nancy A. Healey, BS, Hollace MacGregor, BS, Mheir Doursounian, BS, and Michael A. Zolkewitz, BS Department of Surgery, Brockton/West Rnxburv Veterans Affairs Medical Center; Brigham and Women's Hospital; Harvard Medical School; and the Naval Blood Research Laboratory, Whitaker Cardiovascular Institute, and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts
Background. Platelet dysfunction and increased fibrinolysis are the most important etiologic factors in the hemostatic defect observed f o l l o w i n g the institution of cardiopulmonary bypass. This study examined the effects of heparin per se, administered before the institution of cardiopulmonary bypass, on platelet function and fibrinolysis. Methods. Sampling was perforlned in 55 patients undergoing cardiac operations before and 5 minutes after the routine administration of heparin, beh)re the institution of cardiopulmonary bypass. Results. Heparin administration resulted in a significant prolongation of the bleeding time (from 6.3 - 2.1 to 12.6 + 4.9 minutes; p < 0.00001), a significant reduction in the level of shed blood thromboxane B: (from 1,152 + 669
to 538 + 187 pg/0.1 mL; p = 0.00002), and an increase in the plasma levels of plasmin (from 11.8 + 9.7 to 125.4 + 34.8 U/L; p < 0.0001) and D-dimer (from 571.3 --- 297.1 to 698.5 + 358.6/.tg/mL; p = 0.05). There were no significant differences before and after heparin administration in the plasma levels of fibrinogen, plasminogen, tissue plasminogen activator, antiplasmin, antithrombin I|I, and yon Willebrand factor. Conclusions. Heparin, independent of cardiopulmonary bypass, causes both platelet dysfunction and increased fibrinolysis. The use of an alternative anticoagulant or a lower dose of heparin in conjunction with heparin-coated surfaces might improve the hemostatic balance during open heart operations.
he institution of cardiopulmonary bypass alters heT mostasis and restllts in increased postoperative bleeding [1]. Platelet dysfunction and increased fibrinol-
of the platelet dysfunction observed during and after cardiopulmonary bypass. Hypothermia, for example, has been clearly shown to prolong the bleeding time and to reduce the platelet production of thromboxane A 2 in patients undergoing cardiopulmonary bypass [4]. Heparin, which is universally used in patients undergoing cardiopulmonary bypass, is another extrinsic factor that may influence platelet function in these patients. Heparin has also been shown to have profibrinolytic activity [5]. Hence, heparin may contribute to the hemostatic abnormality of cardiopulmonary bypass not only by its inhibition of thrombin, but also by inducing platelet dysfunction and by promoting fibrinolysis. This investigation was carried out to elucidate the effect of the administration of heparin before the institution of cardiopulmonary bypass on platelet function and fibrinolytic activity in patients undergoing cardiac operations.
ysis are two mechanisms that are primarily responsible for the hemostatic dysfunction induced by cardiopulmonary bypass [11. Although contact with the extracorporeal circuit results in platelet loss secondary to platelet activation, secretion, and degranulation, the resultant thrombocytopenia encountered in the majority of patients u n d e r g o i n g c a r d i o p u l m o n a r y bypass is not severe enough to account for the platelet dysfunction observed in these patients, which is manifested bv a marked prolongation of the postoperative bleeding time [2]. It was postulated that cardiopulmonary bypass induced platelet dysfunction by altering the platelet membrane receptors GPlb and GPllb/llla. A recent collaborative study from out institution demonstrated that the membrane receptors were intact in platelets circulating during cardiopulmonary bypass [3]. It suggested that factors extrinsic to the platelet might be important determinants
~Ann Thorac Sur~, 1995;60:1008-14)
Material and Methods
Patient Population Presented at tile Ihirt~-first .\nnua] kh'etin~t,H [he Societ~ o f ' l h o r a c i c Surgeons, Palm Springs. ( A, lan 30-1 cb [, I~lt~q The o p i n i o n s or assertions cllntaincd hc, rehl art' thilst' ol tilt' authol-~, and are not to be COllHtltlt'd ,'is official ol lellc'cting the vit>~s of the Naxx D e p a r t m e n t or N
O 1995 bv The Societ; ol Th~racic Surgc.~;n~
This study was conducted in 55 patients (54 men, 1 woman) undergoing cardiac operations at the West Roxbury Veterans Affairs Medical Center. The mean age (+ standard deviation) of the patients was 64 + 8.7 years. The operations performed included isolated coronary artery bypass grafting (n = 38), isolated valve replacement (n 5), and valve replacement with coronary artery 0003-4975195159.50 SSDI 0003-4975(95)00668-B
Ann Thorac Surg 1995;60:1008-14
bypass grafting (n 12). All patients signed consent forms approved by the institutional review board. No patient had a history suggestive of an u n d e r l y i n g hemostatic disorder. Patients were not entered into the study if they had a histo D' of aspirin, antiinflammatory, or fibrinolytic drug intake within 1 week before the operation. All patients were premedicated with droperidol and fentanyl citrate (Innovar; Janssen Pharmaceuticals, Piscataway, NJ) and d i p h e n h y d r a m i n e hydrochloride (Benadryl; Parke-Davis, Morris Plains, NJ). Anesthesia was induced with fentanyl and lidocaine and m a i n t a i n e d ~s.ith fentanyl and halothane. After an initial heparin dose of 4 mg/kg, cardiopulmona~; bypass was begun. A membrane oxygenator and a circuit primed with lactated Ringer's solution were used. S u b s e q u e n t heparin dosages were determined according to the activated clotting time, which was maintained greater than 600 seconds. After discontinuation of cardiopulmonary bypass, heparin was neutralized with protamine sulfate given in a ratio of 0.5 mg of protamine to 1.0 mg of the initial heparin dose and 1.0 mg of protamine to 1.0 mg of the s u b s e q u e n t heparin dosages. Heparin reversal was also monitored by the measurement of the activated clotting time.
Blood Samples and Assays After o p e n i n g of the chest and before cannulation for cardiopulmonary bypass, two arterial blood samples were obtained, one before and one 5 minutes after the administration of heparin. Blood was collected for the m e a s u r e m e n t of hematocrit and platelet count employing a Coulter JT Counter (Coulter Electronics, Hialeah, FL). Von Willebrand factor (vWF) antigen concentration was measured with enzvme-linked i m m u n o s o r b e n t assay 16]. The vWF multimer distribution was assessed by separating plasma proteins electrophoretically on a sodium dodecyl sulfate-l% agarose gel using a continuous buffer [71. The vWF antigen was identified by incubating the gel with ~z~I-anti-vWF antibody followed by autoradiography. Each sample was tested at least twice on separate gels to ensure reproducibility. Multimer distribution was assessed by visual inspection of the autoradiograph. Plasminogen and plasmin activity in plasma were measured employing the same substrate [21. Measurements of tissue plasminogen activator were made using enzyme-linked i m m u n o s o r b e n t assay. Samples for m e a s u r e m e n t of fibrinogen were collected in 3.8% sodium citrate tubes and centrifuged at 1,200 g for 10 minutes. After neutralization with protamine, fibrinogen was measured using a Coag-a-Mate X2 photo optical i n s t r u m e n t (General Diagm~stics, O r g a n o n Teknika Corp, Durham, NC). Heparin levels were measured with a chromogenic substrate. D-Dimer, a breakdown product of fibrin, was measured bv an enzyme-linked i m m u n s o r bent assay with a monoclona] antibody [2]. Chromogenic assays were used for the m e a s u r e m e n t of a n t i t h r o m b i n III and antiplasmin.
KHURI ET AL t t E P A R I N EFFECTS O N P L A T E L E T S A N D F1BR1NOLYS1S
1009
Measurement of Bleeding Time and Shed Blood Thromboxane B2 The bleeding time was measured from the lateral aspect of the volar surface of the forearm according to the method of Babson and Babson [8] with the Simplate II bleeding time device (General Diagnostics). With every bleeding time measurement, local skin temperature was recorded by placing a surface skin thermistor (Skin Temperature sensor; Mon-A-Therm, lnc, St. Louis, MO) within a few millimeters of the bleeding time site. Concomitant with bleeding time and temperature measurements, shed blood obtained from the bleeding time site every 30 seconds was aspirated through a b l u n t needle into a tuberculin syringe coated with heparin (1,000 UJmL) and containing 20 txL of ibuprofen for 1 mL of blood (1.9 mg/mL). Each template produced two skin incisions, and two templates were used at each period, one for m e a s u r e m e n t of the duplicate bleeding times a n d the recording of the m e a n value, and the other for collection of the shed blood from the skin incision. A volume of 0.6 mL of blood was collected from the bleeding time site for these measurements. The blood samples were kept on ice until they were centrifuged at 1,650 g (3,000 rpm) in a Sorvall RC-3 centrifuge (DuPont Company, Wilmington, DE) for 10 minutes. The plasma was removed and frozen at 80°C until assays for thromboxane B2 levels were done. Assays were performed with thromboxane B2 (iodine 125) r a d i o i m m u n o a s s a y kits (New England Nuclear Corp, Boston, MA).
l]ah7 Analysis The bleeding time was corrected for temperature by applying the factor described by Valeri and associates 19]. The plasma proteins were corrected for hemodilution as described previously [1l. Comparisons were made between data obtained before and after the administration of heparin using the paired Student's t test. Statistical significance was set at p less than or equal to 0.05. Correlations were performed employing linear regression analysis. Analyses were done using SAS Statistical Software (SAS Institute Inc, Cary, NC). The m e a n values in the narrative and the tables are shown _+ their standard deviation; in the figures, they are shown +_ their standard error.
Results
Skin Temperature, Hematocrit, and Platelet Count The skin temperature at the site of the bleeding time determination drifted downward by an average of 1°C (Fig 1). This drift was ascribed to progressive cooling of the skin secondary to a low a m b i e n t temperature in the operating room and to the o p e n i n g of the chest. In the 55 patients it averaged 29.7 ° = 1.3°C before and 28.7 ° + 1.7°C after heparin administration. This difference, although small, was highly significant (p - 0.0004). The hematocrit before the administration of h e p a r i n was 34.31% : 5.04%. After heparin it was 32.52% + 4.89% (p 0.163). The platelet count before heparin administration in 55 patients ranged from 64,000 to 399,000/tzL. It aver-
1010
KHUR1 El' AL HEPARIN EFFECTS ON PLA[ELETS
33
Ann Thorac Surg 1995;60:1008-14
AND FIBRINOLYSIS
n=55 p=O.O004
\.
n=54 p
n=32 p=O.O0002
3600 -
2O 3000 -
3 c 15
2400-"
1800 -
E ~
29-
<
28-
~
m
5 ooo-
0
26
Heparin
Heparin
Fig 1. Arm skin tempen#un' at tiw ~ite of the bleeding time determination before and 5 minutes ~f?er the administration off heparin in 55 study patienG. The means ~ stamtard error of the mean for eaci~ time point are shown.
aged 212,833 + 66,569//.tL. Five minutes after heparin administration it averaged 198,040 ÷ 56,451//~L (p - 0.24). Concentration
and Activated
Clotting Time
The activated clotting time (ACT) before heparin administration ranged from 105 to 201 seconds and averaged 137.9 +_ 19.2 seconds. In most of the patients, heparin was administered to achieve an ACT of more than 600 seconds. As a result, the ACT after the administration of the first dose of heparin ranged from 479 to 1,000 seconds and averaged 870 + 168 seconds. Heparin concentration in the blood before heparinization was 0.22 +_ 0.11 IU/mL. After the first dose, it ranged from 0.2 to 9.4 1U/mL and averaged 6.26 + 1.53 IU/mL. There was no correlation b et ween the ACT and the blood heparin concentration (r 0.09) after the administration of the first dose of heparin. Platelet Function
Platelet function parameters before and 5 minutes after heparin administration are shown in Table 1. The bleeding time before heparin administration ranged from 3.5 to 10.5 minutes and averaged 6.3 minutes. After the
Table 1. Platelet Function Parameters Bt~ti~re a m t 5 M i n u t e s A f t e r A d m i n i s t r a t i o n o f Heparin
Parameter
n
Before Heparin
After Heparin
p Value
Bleeding time (minutes) Shed blood T x B 2 (pg/0.1 ml) vWF antigen (%)
54
6.3 - 2.1
12.6 - 4.9
0.00001
538 - 187
0.00002
136 " 55
0.64
TxB 2 = thromboxane
32
1152
15 Bz;
-
669
172 r 48 vWF
= vml Willebrand
~-
Pre Heparin
Post
Pre
Heparin
"\\
I..- 10 o? c
factor.
.
o't
Post Heparin
,. Pre
Heparin
Post Heparin
[ i\, 2. Bleeding time and shed blood thromboxane B, in the patients in whom paired measurements were obtained before and 5 minutes after the administration of heparin. The means +_ standard error of the mean for each time point are shown.
administration of heparin, the bleeding time increased in all but 2 patients (Fig 2); it averaged 12.6 minutes. This was a marked and a significant increase c o m p a r e d with the p r e h e p a r i n level (p < 0.00001). The administration of heparin also resulted in a m a r k e d fall in the level of thromboxane B 2 in the blood shed from the site of the bleeding time determination. Paired data were obtained in 32 patients; their results are shown in Table 1 and Figure 2. The m e a n shed blood t h r o m b o x an e B2 level before heparin administration was 11.5 ng/mL. After heparin administration, it fell to 5.5 n g / m L (p - 0.00002). There was a moderate but significant negative correlation between the bleeding time and the shed blood thromboxane B2 (r = -0.43; p < 0.0005). There were no significant negative correlations b e t w e e n the skin t e m p e r a t u r e and the changes in the corrected bleeding time and the shed blood t h r o m b o x an e B2 level. Therefore, the changes in the bleeding time and t h r o m b o x an e B2 were not due to the slight fall in skin t e m p e r a t u r e that was observed between the preheparin and postheparin time points. Von Willebrand factor antigen concentration was measured before and 5 minutes after heparin administration in 15 patients. As shown in Table 1, the vWF antigen concentration before and after heparin were not significantly different. The vWF m u l t i m e r distribution was not altered in patients after infusion of heparin, as m e a s u r e d by visual inspection of vWF antigen on autoradiographs (Fig 3). Fibrinolytic Activity
Table 2 displays the results of fibrinolytic and related parameters before and after the administration of heparin. Plasmin was m e a s u r e d in 19 patients. As shown in Figure 4, heparin administration resulted in a tenfold increase in plasmin from a p r e h e p a r i n average of 11.8 U / L to a postheparin average of 125.4 U/L (p < 0.0001). In
Ann Thorac Surg 1995;60:1008 14
KHURI ET AL HEPARIN EFFECTSON PLATELETSAND FIBRINOLYSIS
200
n=16 p< 0001
1011
n=51 p=O.05 1500-
150
~
/
,oo
_ ,ooo
t
~ ,5oj
50 11/
250
0 A
N
B
A
1
B
2
A
B
3
Pat i en t Fi,~,,3. Autoradio<\,raph of yon Wilh'br, nzd tactor in normal pool plasma (N), rout.from 3 retu'esclttativt' patients q/1,5,b
these patients, the preheparin and postheparin administration levels of plasmin correlated significantly with the preheparin and postheparin bleeding times (r 0.75; p 0.005) (Fig 5). In 51 patients in whom D-dimer measurements were made, the administration of heparin elicited an increase in D-dimer level in 43 (77%) and no change or a decrease in 12 (23",h) (see Fig 4). On the average, there was a modest but significant increase in the D-dimer level from a preheparin mean of 571.3 ~g/mL to a postheparin mean of 698.5/~g/mL (p 0.05). As shown in Table 2, the administration of heparin elicited no significant changes in the plasma levels of fibrinogen, tissue plasminogen activator, plasminogen, antiplasmin, and antithrombin It1.
Comment
Parameter
n 16 51 25 28
9.7 571.3 297.1 7.12 5.1 387.7 84. I 11,H
+
125.4 " 698.5 : 6.47 372.8
34.8 I' 358.6h 5.17I" 109.1l'
Posl Heparin
Plateh't D y s f u n c t i o n in O p e n Heart Operations and the Eft-cot o f H e p a r i n It has long been recognized that patients u n d e r g o i n g cardiac operations have increased postoperative bleeding. This has been attributed to a cardiopulmonary byp a s s - i n d u c e d hemostatic defect, which was thought to be
175
150 '
i I
Value O.OI)(ll 0.05
j
125 -i
I'
~100
Plasmin (U/L) D-dimer (/xglmL) t-PA (nglmL) Fibrinogen (mg/dL) b Plasminogen (",) Antiplasmin C,,) Antithrombin 111 (%,)
t
Pre Heparin
before the institution of cardiopulmonary bypass, causes platelet dysfunction (manifested by a prolongation of the bleeding time and a reduction in the ability of the platelet to produce thromboxane A z in vivo) and increased fibrinolvsis (manifested by increased plasma levels of plasmin and o-dimer). Hence, heparin contributes to the hemostatic defect observed in patients u n d e r g o i n g cardiac operations not only by inhibiting coagulation through its effect on a n t i t h r o m b i n III, but also by eliciting direct adverse effects on the platelet and the fibrinolytic system.
Lllllt" 2. Parameters Related to Fibrinolysis Before amt 3 Minutes Afitcr Administration of Hcparin
,After Heparin
%st Heparin
lik~4. Plasma h'vels of plasmin and u-dimer in the patients in whom paired measurements were obtained before and 5 minutes l{fter the administration qf heparin. The means - standard error of the mean for each time point are shown.
This study demonstrates that, in patients undergoing open heart operations, the administration of heparin,
Before 1teparin
o [
Pre Heparin
E
jJ~
-
"
I
75 -4
•
NS NS
50
NS XS NS
25
i
/O'"
,.'J
•
r = 0.75
p = 0,005
19 19 28
79.3 75.2 SO
I2.~q 8.4 l2
84 71 8(I
14b 21h 12I>
[.. 1
••
••
r
~
3
5
~
7
9
~
11
r
q
T
i
i
13
15
17
19
21
Bleeding Time (minutes) "~ \"alue
NS
corrected
Inr d i ] u t h l n ,
I]Ot S i g l l i t i c a n l ;
l-P\
h Sdill[~lt'- n e u l r a l i / e d w i t h prlil,lnlint,. [iSSLIC t~l
Fi~,,5. Correlation between bleedit g tinw and plasma plasmin levels l,ctbrc and ,5 minutes after the administration of heparin.
1012
KHURI ET AL HEPARIN EFFECTS ON PLAH 1.111S ;\NI) FIBRINOI.YSIS
due mostly to platelet dysfunction and, to a lesser extent, increased fibrinolysis [1, 101. Because the magnitude of thrombocytopenia encountered in the course of a cardiac operation is not severe enough to account for the marked extension of the bleeding time observed during and after cardiopulmonaD ~ bypass [2], investigations in this field have focused on specific platelet abnormalities that might be induced by cardiopulmonary bypass. The platelet defect of cardiopulmonary bypass has been postulated to be due to a loss of the platelet membrane receptors responsible for platelet adhesion and aggregation. Loss of platelet m e m b r a n e receptors for both the vWF and fibrinogen were reported during and after cardiopulmonary bypass II1-13]. However, the methods with which the platelets were prepared in these studies involved centrifugation and gel filtration of the platelets before assay, thereby introducing the possibility of an artifactual in vitro decrease in platelet surface GPIb-IX complex (the vWF receptor) and GPllb-llla complex (the fibrinogen receptor) as a result of proteolysis or activation. In a study of 20 patients undergoing cardiac operations, we used a flow cytometric method that allowed us to study the platelet GP[b-IX and GPllb/llla in whole blood, thereby avoiding potential artefactual reductions in platelet surface glycoprotein receptors. With this method, we demonstrated that cardiopulmona D, bypass did not result in a decrease in the platelet surface expression of either the GPIb-IX complex or the GPllbIlia complex [3]. This study underscored the possibility of factors extrinsicto the platelet itself that might contribute to the platelet dysfunction observed in patients undergoing cardiac operations. Two such factors are hypothermia and heparin. In a study of 37 patients undergoing cardiopulmonary bypass, we confirmed the adverse effect of hypothermia on platelet function by demonstrating that hypothermia resulted in a significant prolongation of the bleeding time and a significant reduction in the ability of the platelet to produce thromboxane A 2 [4[. Platelet function in the present study was assessed bv the measurement of the bleeding time and the level of thromboxane B 2 in the blood shed from the skin at the site of the bleeding time determination. Despite its limitations, the bleeding time continues to be a reliable indicator of platelet function and platelet-vessel wall interaction [1]. The measurement of the shed blood thromboxane B 2 has also been shown to be a reliable indicator of platelet function [2, 14]. It has the distinct advantage of reflecting in vivo platelet function, making it an ideal measurement for use in patients undergoing cardiopulmonary bypass [2-4]. Both measurements indicated a marked reduction in platelet function after the administration of heparin. Although it has been long recognized that heparin caused a prolongation of the bleeding time [15], its recognition as an etiologic factor in postbypass hemorrhage is only recent [1, 161. The mechanism with which heparin might cause platelet dysfunction was partially addressed in our previous study in which whole blood flow cytometry was employed in the characterization of the platelet membrane glycoproteins Ib and IIb-llIa and platelet activation be-
Ann Thorac Surg 1995;60:1008-14
fore and after cardiopulmonary bypass [3]. That study provided evidence that heparin suppressed platelet activation in vivo via inhibition of endogenousthrombin, the latter being the most important platelet agonist in vivo. Another possible mechanism for the inhibition of platelet function by heparin is through an inhibition of vWF activity. Ristocetin is a cationic antibiotic that induces in vitro binding of vWF to its receptor on platelet GPlb. Sobel and associates [17] demonstrated that the intravenous administration of heparin to patients before open heart operations reduced ristocetin cofactor activity by 58%. This impairment of vWF-dependent platelet function was closely related to plasma heparin levels but not to plasma vWF levels. It appeared to be caused by direct binding of heparin to vWF in solution, but may also be a consequence of heparin binding directly to the platelet surface [18]. In the present study, we have demonstrated that the concentration and multimer distribution of the vWF were unaffected by the administration of heparin. Hence our data are consistent with the hypothesis that heparin reduces platelet function by preventing the vWF from binding to its receptor on the GPlb on the platelet. The third mechanism by which heparin can induce platelet dysfunction is through its effect on plasmin. This is explained in the section below.
Fibrinolysis During Cardiopulmonary Bypass and the Effect of Heparin There are a number of pathways that could lead to increased fibrinolysis during cardiopulmonary bypass operations, including the activation of kallikrein and the release of tissue plasminogen activator from the endothelial cell [1[. Fibrin(ogen) degradation products and D-dimer levels increase during and following cardiopulmonary bypass ]1, 19]. Administration of aprotinin during cardiopulmonary bypass prevents the formation of fibrin degradation products and the reduction in %antiplasmin activity [20]. It also reduces the bleeding time ]21[. These observations, the mechanism of action of aprotinin [22], and the clinical effectiveness of aprotinin in decreasing blood loss ]21[, strongly suggest that fibrinolysis is important in the hemorrhagic diathesis associated with cardiopulmonary bypass operations. The present study demonstrates that, in patients before the institution qf cardiopulmona~ bypass, the administration of heparin increases fibrinolysis as evidenced by a uniform increase in plasmin and a partial but significant increase in D-dimer level. Heparin and its fractions, however, have been known to possess profibrinolytic activities for more than a decade [5]. Fareed and associates [5] reported an increase in tissue plasminogen activator levels in h u m a n volunteers after the institution of intravenous and subcutaneous heparin over a period of 10 days (daily dose of 7,500 units). They postulated a n u m b e r of profibrinolytic actions of heparin and its fractions. Several recent studies have shown that heparin and heparin-like compounds stimulated cell surface plasminogen activation by 10- to 17-fold [23]. Because this interaction occurred at the cell surface level, it would be unlikely that
Ann Thorac Surg 1995;60:1{)1)8 14
the profibrinolytic effect of heparin would be reflected bv the levels of plasminogen in the plasma. Hence, in our study, we did not show anv change in the plasma levels of plasminogen with the administration of heparin. The lack of a significant increase in tissue plasminogen activator after the administration of heparin did not denote a lack of profibrinolytic activity because the plasma level tissue plasminogen activator antigen may not correlate with the functional activity of the molecule in patients u n d e r g o i n g cardiopulmonary bypass. The rise in plasmin and o-dimer levels after the administration of heparin indicated an increase in profibrinolytic activity. It is of note that plasmin, fibrin degradation products, and Ddimer have all been shown to interfere with platelet aggregation, p r e s u m a b l y through a proteolytic effect on the platelet GPI|Ia in the case of plasmin [24] and by competing with fibrinogen for GPIIb/llIa b i n d i n g in the case of the degradation products. Hence the generation of plasmin, stimulated bv heparin, provides another possible mechanism by which heparin impairs platelet function as well. A confirmatory finding of this mechanism is the significant correlation that was observed in this study between the bleeding time and the plasma levels of plasmin.
Clinical Implications The hemostatic defect observed in patients undergoing cardiac operations is not fullv reversed with the administration of protamine and the discontinuation of cardiop u l m o n a r y bypass. In fact, D-dimer levels increase after the administration of protamine, and the bleeding time continues to be significantly elevated in the hours after the discontinuation of cardiopulmona D, bypass [1, 2]. The bleeding time at 2 hours after cardiopulmona D' bypass was shown to be an important d e t e r m i n a n t of postoperative blood loss [2]. Interventions aimed at reducing postoperative platelet dvsfunction and fibrinolysis are likely to reduce postoperative blood loss in patients u n d e r g o i n g cardiac operations. Because a variety of complex factors could contribute to the hemostatic defect observed in the initial postoperative hours in these patients, it is very difficult to appreciate the specific contribution of the administration of heparin per se to this defect. Nevertheless, the present study raises questions as to whether an alternative anticoagulant or a lower dose of heparin might improve the hemostatic balance during open heart operations. In the present study, the ACT was kept on the high side to achieve full suppression of thrombin. Significantly lower ACT levels have been employed in conjunction with heparin-coated surfaces. It remains to be determined whether combining low-dose heparin with heparin-coated surfaces would suppress thrombin adequately and, at the same time, reduce the adverse effect of heparin on platelet function a n d fibrinolysis. It also r e m a i n s to be d e t e r m i n e d whether an alternative anticoagulant that would not interfere with either the platelet function or the fibrinolytic process can improve postoperative hemostasis in the cardiac surgical patient.
KHURIET AL HFPARIN EFFF,CTS ON PLATELETSAND FIBRtNOLYSIS
1013
We acknowledge the valuable help of Jennifer Marjani and Frank Glavin in the preparation of the manuscript. Supported by the US Navy (Office of Naval Research Contract N00014-94-C-0149 with funds provided by the Naval Medical Research and Development Command), and by the Richard Warren Surgical Research and Educational Fund, Westwood, Massachusetts.
References I. Khuri SF, Michelson AD, Valeri CR. The effect of cardiopulmonary bypass on hemostasis and coagulation. In: Loscalzo J, Schafer AI, eds. Thrombosis and hemorrhage. Cambridge, MA: Blackwell Scientific, 1994:1051-73. 2. Khuri SF, Wolfe JA, Josa M, et al. Hematologic changes during and after cardiopulmonary bypass and their relationship to the bleeding time and nonsurgical blood loss. J Thorac Cardiovasc Surg 1992;104:94-107. 3. Kestin AS, Valeri CR, Khuri SF, et al. The platelet function defect of cardiopulmonary bypass. Blood 1993;82:107-17. 4. Valeri CR, Khabbaz K, Khuri SF, et al. Effect of skin temperature on platelet function in patients undergoing extracorporeal bypass. J Thorac Cardiovasc Surg 1992;104:108-16. 5. Fareed J, Walenga JM, Hoppensteadt DA, Messmore HL. Studies on the profibrinolytic actions of heparin and its fractions. Semin Thromb Hemost 1985;11:199-207. 6. Pennv W, Weinstein MJ, Ware JA, Salzman E. Correlation of circuiating yon Willebrand factor levels with cardiovascular henlodynamics. Circulation 1991;83:1630-6. 7. Weinstein MJ, Ware JA, Troll J, Salzrnan EW. Changes in yon Willebrand factor during cardiac surgery: effect of desmopressin acetate. Blood 1988;71:1648-55. 8. Babson SR, Babson AL. Development and evaluation of a disposable device for performing simultaneous duplicate bleeding time determinations. Am J Clin Pathol 1978;70: 406- 8. 9. Valeri CR, MacGregor H, Pompei F, Khuri SF. Acquired abnormalities of platelet function. [Letter]. N Engl J Med 1991;324:1670. 10. Woodman RC, Harker LA. Bleeding complications associated with cardiopulmonary bypass. Blood 1990;76:1680-97. II. Rinder CS, Mathew JP, Rinder HM, Bonan J, Ault KA, Smith BR. Modulation of platelet surface adhesion receptors during cardiopulmonary bypass. Anesthesiology 1991;75:563-70. 12. Van Oeveren W, Harder MP, Roozendaal KJ, Eijsman L, Wildevuur CRH. Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:788-96. 13. Wenger RK, Lukasiewicz H, Mikuta BS, Niewiarowski S, Edmunds LH. Loss of platelet fibrinogen receptors during clinical cardiopulmonary bypass. J Thorac Cardiovasc Surg 1989;97:235-9. 14. Gerrard JM, Tavback S, Singhroy S, et al. In vivo measurement {}f thromboxane B2 and 6-keto-prostaglandin F1.~ in humans in response to a standardized vascular injury and the influence of aspirin. Circulation 1989;79:29-38. 15. Heiden D, Mielke CH, Rodvien R. Impairment by heparin of primary haemostasis and platelet (C14) 5-hydroxytryptamine release. Br J Haematol 1977;36:427-36. 16. John LCH, Rees GM, Kovacs lB. Inhibition of platelet function b~ heparin. J Thorac Cardiovasc Surg 1993;105:816-22. 17. Sobel M, McNeill PM, Carlson PL, et al. Heparin inhibition of yon Willebrand factor-dependent platelet function in vitro and in vivo. J Clin Invest 1991;87:1787-93. 18. Horne MK Ill, Chao ES. Heparin binding to resting and activated platelets. Blood 1989;74:238-43. 19. Giuliani R, Szwarcer E, Martinez Aquino E, Palumbo G. Fibrin-dependent fibrinolytic activity during extracorporeal circulation. Thromb Res 1991;61:369-73. 2(}. Blauhut B, Gross C, Necek S, Doran JE, Spath P, Lundsgaard-]tansen P. Effects of high-dose aprotinin on blood
1014
KHURI E~f AL HEPARIN EFFECTS ON PLATEI]{IS ANI) FIBRINOLYSIS
loss, platelet function, fibrinolysis, c o m p l e m e n t , a n d renal function after c a r d i o p u l m o n a r y b y p a s s . J T h o r a c C a r d i o v a s c S u r g 1991;101:958- 67. 21. B i d s t r u p BP, Royston D, Sapsford RN, 1-aylor KM. R e d u c t i o n in blood loss a n d blood u s e after c a r d i o p u l m o n a r y b y p a s s with h i g h d o s e aprotinin. J Thorac C a r d i o v a s c S u r g 1989;97: 364-72. 22. M a n n u c c i PM. N o n t r a n s f u s i o n a l modalities. In: Loscalzo ],
Ann Thorac Surg 1995;60:1008-14
Schafer AI, eds. T h r o m b o s i s a n d h e m o r r h a g e . C a m b r i d g e , MA: Blackwell Scientific, 1994:1117-35. 23. Rijken DC, de M u n k GA, Jie AF. i n t e r a c t i o n s of p l a s m i n o g e n activators a n d p l a s m i n o g e n with heparin: effect of ionic s t r e n g t h . T h r o m b H a e m o s t 1993;70:867-72. 24. O u i m e t H, Loscalzo J. Reciprocating autocatalytic interactions b e t w e e n platelets a n d the p l a s m i n o g e n activation s y s t e m . T h r o m b Res (in press).
DISCUSSION D R REPHAEL M O H R ( T e I - H a s h o m e r , Israel}: In our experience we e v a l u a t e d t h e effect of v a r i o u s s t a g e s of p r e b y p a s s h e p a r i n a d m i n i s t r a t i o n a n d so on on platelet function with v a r i o u s m e t h o d s , i n c l u d i n g in vitro, s t a n d a r d PLP aggregation, a n d w h o l e blood a g g r e g a t i o n , a n d we also e v a l u a t e d it on the r e s p o n s e of the blood from t h e patients to the extracellular matrix m o d e l as v i e w e d by electron microscopy. In all t h o s e s t u d i e s t h e initial plalelet d y s f u n c t i o n o b s e r v e d ~ as always after initiation of c a r d i o p u l m o n a D, b y p a s s a n d the levels of d y s f u n c tion were a l m o s t always o b s e r v e d w h e n b y p a s s t e m p e r a t u r e w e n t down. However, w h e n e v e r we m e a s u r e d the effect of h e p a r i n a d m i n i s t r a t i o n on platelet ftmction with all t h e s e rnodels, we n e v e r were able to d e m o n s t r a t e w h a t yotl just s h o w e d us, that platelet function is in a n y w a v d e r a n g e d by h e p a r i n a d m i n istration. How can w~u explain this discrepancy? D R KHURI: O t h e r investigators, e m p l o y i n g various m e t h o d o l ogies, h a v e also s h o w n recently that h e p a r i n c a u s e s significant platelet dysfunction. In this s t u d y we have u s e d a very reliable m e a s u r e of in vivo platelet function, w h i c h is the ability of the platelet to p r o d u c e t h r o m b o x a n e m r e s p o n s e to a b l e e d i n g insult. I can only explain the d i s c r e p a n c y b e t w e e n o u r findings a n d y o u r s on the basis of possible m e t h o d o l o g i c differences. D R S A F U H A T T A R {Baltimore, MD): 1 c o n g r a t u l a t e Dr Khuri a n d his associates on a w e l l - d e s i g n e d study. In the early 1970s 1 p r e s e n t e d a p a p e r on the effects of h e p a r i n on platelets at the IVth International C o n g r e s s on T h r o m b o s i s a n d H e m o s t a s i s . A g g r e g a t i o n w a s i n d u c e d by h e p a r i n tested in platelet-rich p l a s m a . The effect of h e p a r i n on platelet a g g r e g a tion was f o u n d to be proportional to the c o n c e n t r a t i o n ot h e p a r i n . We also c o m p a r e d the effect of intestinal h e p a r i n v e r s u s l u n g heparin; there w a s a significant increase in the a g g r e g a t i o n of the platelets w h e n intestinal h e p a r i n w a s u s e d in c o m p a r i s o n with t h e l u n g heparin. T h e r e a s o n is that h e p a r i n is a h e t e r o g e n e o u s s u b s t a n c e that p r o b a b l y has bits of intestines, spleen, a n d o t h e r t h i n g s in it; therefore, t h e s e t i s s u e s are the o n e s that p r o d u c e the platelet aggregation. My q u e s t i o n s to Dr Khuri are the following. First, did vou u s e t h e platelet c o u n t as a function? T h e c o u n t of the platelets does not m e a n very m u c h ; the platelets m a v be f u n c t i o n l e s s a n d vet t h e c o u n t m a y be all right. Second, w h a t kind of h e p a r i n did you use, intestinal or lung h e p a r i n ? Third, if you use a m o r e purified h e p a r i n , will it m i n i m i z e the effects of h e p a r i n on the platelets? D R L. HENRY E D M U N D S (Philadelphia, PA): Doctor Khuri, 1 e n j o y e d y o u r p a p e r a n d l enjoyed the abstract, a n d 1 a m familiar with y o u r work with Drs Kestin a n d Mickelson. [ t e p a r i n is a w e l l - k n o w n a g o n i s t for platelets, b u t also it is a less well-kno~x n a g o n i s t for n e u t r o p h i l s a n d c o m p l e m e n t . It is certainly not an ideal a n t i c o a g u l a n t by a long shot. O u r g r o u p believes that t h e r e is an e,xtrinsi¢ factor that s u p p r e s s e s platelet function, as w m r g r o u p h a s p o i n t e d out. However, I have to ask you w h e t h e r you
think t h e r e is an intrinsic defect p r o d u c e d by c a r d i o p u l m o n a r y b y p a s s in platelets, as o u r data lead u s to believe. I a m d i s t u r b e d by y o u r c o n c l u d i n g c o m m e n t , that m a y b e a c o m b i n a t i o n of s u r f a c e - b o u n d h e p a r i n a n d s o m e o t h e r anticoa g u l a n t will get us a r o u n d this p r o b l e m . R e c o m b i n a n t h i r u d i n is the alternative a n t i c o a g u l a n t that is b e i n g c o n s i d e r e d the most. This is a v e D' t i g h t - b i n d i n g inhibitor of t h r o m b i n , b u t it does n o t p r e v e n t the h~rmation of t h r o m b i n . It is i m p o r t a n t for e v e r y o n e w h o does cardiac s u r g e r y to realize that t h r o m b i n is f o r m e d in progressively i n c r e a s i n g a m o u n t s d u r i n g every, c a r d i o p u l m o narv b y p a s s operation, a n d that it is a d a n g e r o u s e n z y m e to circulate. So the idea of u s i n g s u r f a c e - b o u n d h e p a r i n with a direct t h r o m b i n inhibitor s u c h as h i r u d i n h a s little a p p e a l to m e . S u r f a c e - b o u n d h e p a r i n d o e s n o t a p p e a r to h a v e an a n t i c o a g u lant effect a n d therefore is unlikely to help. W o u l d y o u c o m m e n t on w h e t h e r you believe, or y o u r g r o u p believes, that t h e r e is an intrinsic defect in platelets p r o d u c e d by cardiopulmonary bypass? DR KHURI: T h a n k you, Dr E d m u n d s . 1 a m very appreciative of v~mr c o m m e n t s . I can very well u n d e r s t a n d y o u r c o n c e r n a b o u t m y r e m a r k r e g a r d i n g the possible a d v a n t a g e of c o m b i n i n g low-dose h e p a r i n with h e p a r i n - c o a t e d circuits, a n d 1 look forward to the publication of y o u r data which, as I i n t e r p r e t f r o m w m r c o m m e n t s , will s h o w that s u c h a c o m b i n a t i o n w o u l d n o t a d e q u a t e l y p r e v e n t the f o r m a t i o n of t h r o m b i n , which, 1 agree, is a molecule we n e e d to avoid d u r i n g b y p a s s . In the p r e s e n t study, we m a i n t a i n e d relatively h i g h activated clotting time levels (an a v e r a g e of 700 to 800 s e c o n d s ) to e n s u r e specifically that t h e t h r o m b i n was a d e q u a t e l y neutralized. As to the q u e s t i o n of w h e t h e r the c a r d i o p u l m o n a r y b y p a s s i n d u c e d platelet defect is intrinsic or extrinsic to the platelet, o u r s t u d i e s to date h a v e identified h y p o t h e r m i a a n d h e p a r i n as extrinsic factors r e s p o n s i b l e , in part, for this platelet d y s f u n c tion. O u r s t u d i e s h a v e not r u l e d o u t the p r e s e n c e of a cardiop u l m o n a r y b y p a s s - i n d u c e d intrinsic platelet defect. As m y coll e a g u e s a n d 1 have p u b l i s h e d in Blood, however, the m e c h a n i s m of this intrinsic defect is not likely to be a loss of t h e platelet m e m b r a n e receptors GPIb a n d GPlIb-IIla, as earlier s t u d i e s h a d suggested. Doctor Attar, we appreciate the fact that y o u were w a y a h e a d of your time in s h o w i n g , m o r e t h a n 20 y e a r s ago, that h e p a r i n c a u s e d a b n o r m a l platelet aggregation. W e h a v e not u s e d platelet a g g r e g a t i o n in the a s s e s s m e n t of platelet function b e c a u s e of the variability in that t e c h n i q u e . Instead, we h a v e o p t e d for t h e m viw~ test of m e a s u r i n g t h r o m b o x a n e B2 in t h e blood s h e d at the site of the b l e e d i n g time d e t e r m i n a t i o n . W e believe t h a t this test of platelet f u n c t i o n is m o r e applicable a n d r e l e v a n t to t h e patient u n d e r g o i n g c a r d i o p u h n o n a r y b y p a s s . M a y b e that is w h y t h e r e is a d i s c r e p a n c y b e t w e e n o u r data a n d t h o s e of Dr M o h r from Israel. As to y o u r specific q u e s t i o n s a b o u t h e p a r i n , we h a v e u s e d porcine h e p a r i n , a n d we have h a d no experience with t h e m o r e purified f o r m s of heparin.
Background. Platelet dysfunction and increased fibrinolysis are the most important etiologic factors in the hemostatic defect observed f o l l o w i n g the institution of cardiopulmonary bypass. This study examined the effects of heparin per se, administered before the institution of cardiopulmonary bypass, on platelet function and fibrinolysis. Methods. Sampling was perforlned in 55 patients undergoing cardiac operations before and 5 minutes after the routine administration of heparin, beh)re the institution of cardiopulmonary bypass. Results. Heparin administration resulted in a significant prolongation of the bleeding time (from 6.3 - 2.1 to 12.6 + 4.9 minutes; p < 0.00001), a significant reduction in the level of shed blood thromboxane B: (from 1,152 + 669
to 538 + 187 pg/0.1 mL; p = 0.00002), and an increase in the plasma levels of plasmin (from 11.8 + 9.7 to 125.4 + 34.8 U/L; p < 0.0001) and D-dimer (from 571.3 --- 297.1 to 698.5 + 358.6/.tg/mL; p = 0.05). There were no significant differences before and after heparin administration in the plasma levels of fibrinogen, plasminogen, tissue plasminogen activator, antiplasmin, antithrombin I|I, and yon Willebrand factor. Conclusions. Heparin, independent of cardiopulmonary bypass, causes both platelet dysfunction and increased fibrinolysis. The use of an alternative anticoagulant or a lower dose of heparin in conjunction with heparin-coated surfaces might improve the hemostatic balance during open heart operations.
he institution of cardiopulmonary bypass alters heT mostasis and restllts in increased postoperative bleeding [1]. Platelet dysfunction and increased fibrinol-
of the platelet dysfunction observed during and after cardiopulmonary bypass. Hypothermia, for example, has been clearly shown to prolong the bleeding time and to reduce the platelet production of thromboxane A 2 in patients undergoing cardiopulmonary bypass [4]. Heparin, which is universally used in patients undergoing cardiopulmonary bypass, is another extrinsic factor that may influence platelet function in these patients. Heparin has also been shown to have profibrinolytic activity [5]. Hence, heparin may contribute to the hemostatic abnormality of cardiopulmonary bypass not only by its inhibition of thrombin, but also by inducing platelet dysfunction and by promoting fibrinolysis. This investigation was carried out to elucidate the effect of the administration of heparin before the institution of cardiopulmonary bypass on platelet function and fibrinolytic activity in patients undergoing cardiac operations.
ysis are two mechanisms that are primarily responsible for the hemostatic dysfunction induced by cardiopulmonary bypass [11. Although contact with the extracorporeal circuit results in platelet loss secondary to platelet activation, secretion, and degranulation, the resultant thrombocytopenia encountered in the majority of patients u n d e r g o i n g c a r d i o p u l m o n a r y bypass is not severe enough to account for the platelet dysfunction observed in these patients, which is manifested bv a marked prolongation of the postoperative bleeding time [2]. It was postulated that cardiopulmonary bypass induced platelet dysfunction by altering the platelet membrane receptors GPlb and GPllb/llla. A recent collaborative study from out institution demonstrated that the membrane receptors were intact in platelets circulating during cardiopulmonary bypass [3]. It suggested that factors extrinsic to the platelet might be important determinants
~Ann Thorac Sur~, 1995;60:1008-14)
Material and Methods
Patient Population Presented at tile Ihirt~-first .\nnua] kh'etin~t,H [he Societ~ o f ' l h o r a c i c Surgeons, Palm Springs. ( A, lan 30-1 cb [, I~lt~q The o p i n i o n s or assertions cllntaincd hc, rehl art' thilst' ol tilt' authol-~, and are not to be COllHtltlt'd ,'is official ol lellc'cting the vit>~s of the Naxx D e p a r t m e n t or N
O 1995 bv The Societ; ol Th~racic Surgc.~;n~
This study was conducted in 55 patients (54 men, 1 woman) undergoing cardiac operations at the West Roxbury Veterans Affairs Medical Center. The mean age (+ standard deviation) of the patients was 64 + 8.7 years. The operations performed included isolated coronary artery bypass grafting (n = 38), isolated valve replacement (n 5), and valve replacement with coronary artery 0003-4975195159.50 SSDI 0003-4975(95)00668-B
Ann Thorac Surg 1995;60:1008-14
bypass grafting (n 12). All patients signed consent forms approved by the institutional review board. No patient had a history suggestive of an u n d e r l y i n g hemostatic disorder. Patients were not entered into the study if they had a histo D' of aspirin, antiinflammatory, or fibrinolytic drug intake within 1 week before the operation. All patients were premedicated with droperidol and fentanyl citrate (Innovar; Janssen Pharmaceuticals, Piscataway, NJ) and d i p h e n h y d r a m i n e hydrochloride (Benadryl; Parke-Davis, Morris Plains, NJ). Anesthesia was induced with fentanyl and lidocaine and m a i n t a i n e d ~s.ith fentanyl and halothane. After an initial heparin dose of 4 mg/kg, cardiopulmona~; bypass was begun. A membrane oxygenator and a circuit primed with lactated Ringer's solution were used. S u b s e q u e n t heparin dosages were determined according to the activated clotting time, which was maintained greater than 600 seconds. After discontinuation of cardiopulmonary bypass, heparin was neutralized with protamine sulfate given in a ratio of 0.5 mg of protamine to 1.0 mg of the initial heparin dose and 1.0 mg of protamine to 1.0 mg of the s u b s e q u e n t heparin dosages. Heparin reversal was also monitored by the measurement of the activated clotting time.
Blood Samples and Assays After o p e n i n g of the chest and before cannulation for cardiopulmonary bypass, two arterial blood samples were obtained, one before and one 5 minutes after the administration of heparin. Blood was collected for the m e a s u r e m e n t of hematocrit and platelet count employing a Coulter JT Counter (Coulter Electronics, Hialeah, FL). Von Willebrand factor (vWF) antigen concentration was measured with enzvme-linked i m m u n o s o r b e n t assay 16]. The vWF multimer distribution was assessed by separating plasma proteins electrophoretically on a sodium dodecyl sulfate-l% agarose gel using a continuous buffer [71. The vWF antigen was identified by incubating the gel with ~z~I-anti-vWF antibody followed by autoradiography. Each sample was tested at least twice on separate gels to ensure reproducibility. Multimer distribution was assessed by visual inspection of the autoradiograph. Plasminogen and plasmin activity in plasma were measured employing the same substrate [21. Measurements of tissue plasminogen activator were made using enzyme-linked i m m u n o s o r b e n t assay. Samples for m e a s u r e m e n t of fibrinogen were collected in 3.8% sodium citrate tubes and centrifuged at 1,200 g for 10 minutes. After neutralization with protamine, fibrinogen was measured using a Coag-a-Mate X2 photo optical i n s t r u m e n t (General Diagm~stics, O r g a n o n Teknika Corp, Durham, NC). Heparin levels were measured with a chromogenic substrate. D-Dimer, a breakdown product of fibrin, was measured bv an enzyme-linked i m m u n s o r bent assay with a monoclona] antibody [2]. Chromogenic assays were used for the m e a s u r e m e n t of a n t i t h r o m b i n III and antiplasmin.
KHURI ET AL t t E P A R I N EFFECTS O N P L A T E L E T S A N D F1BR1NOLYS1S
1009
Measurement of Bleeding Time and Shed Blood Thromboxane B2 The bleeding time was measured from the lateral aspect of the volar surface of the forearm according to the method of Babson and Babson [8] with the Simplate II bleeding time device (General Diagnostics). With every bleeding time measurement, local skin temperature was recorded by placing a surface skin thermistor (Skin Temperature sensor; Mon-A-Therm, lnc, St. Louis, MO) within a few millimeters of the bleeding time site. Concomitant with bleeding time and temperature measurements, shed blood obtained from the bleeding time site every 30 seconds was aspirated through a b l u n t needle into a tuberculin syringe coated with heparin (1,000 UJmL) and containing 20 txL of ibuprofen for 1 mL of blood (1.9 mg/mL). Each template produced two skin incisions, and two templates were used at each period, one for m e a s u r e m e n t of the duplicate bleeding times a n d the recording of the m e a n value, and the other for collection of the shed blood from the skin incision. A volume of 0.6 mL of blood was collected from the bleeding time site for these measurements. The blood samples were kept on ice until they were centrifuged at 1,650 g (3,000 rpm) in a Sorvall RC-3 centrifuge (DuPont Company, Wilmington, DE) for 10 minutes. The plasma was removed and frozen at 80°C until assays for thromboxane B2 levels were done. Assays were performed with thromboxane B2 (iodine 125) r a d i o i m m u n o a s s a y kits (New England Nuclear Corp, Boston, MA).
l]ah7 Analysis The bleeding time was corrected for temperature by applying the factor described by Valeri and associates 19]. The plasma proteins were corrected for hemodilution as described previously [1l. Comparisons were made between data obtained before and after the administration of heparin using the paired Student's t test. Statistical significance was set at p less than or equal to 0.05. Correlations were performed employing linear regression analysis. Analyses were done using SAS Statistical Software (SAS Institute Inc, Cary, NC). The m e a n values in the narrative and the tables are shown _+ their standard deviation; in the figures, they are shown +_ their standard error.
Results
Skin Temperature, Hematocrit, and Platelet Count The skin temperature at the site of the bleeding time determination drifted downward by an average of 1°C (Fig 1). This drift was ascribed to progressive cooling of the skin secondary to a low a m b i e n t temperature in the operating room and to the o p e n i n g of the chest. In the 55 patients it averaged 29.7 ° = 1.3°C before and 28.7 ° + 1.7°C after heparin administration. This difference, although small, was highly significant (p - 0.0004). The hematocrit before the administration of h e p a r i n was 34.31% : 5.04%. After heparin it was 32.52% + 4.89% (p 0.163). The platelet count before heparin administration in 55 patients ranged from 64,000 to 399,000/tzL. It aver-
1010
KHUR1 El' AL HEPARIN EFFECTS ON PLA[ELETS
33
Ann Thorac Surg 1995;60:1008-14
AND FIBRINOLYSIS
n=55 p=O.O004
\.
n=54 p
n=32 p=O.O0002
3600 -
2O 3000 -
3 c 15
2400-"
1800 -
E ~
29-
<
28-
~
m
5 ooo-
0
26
Heparin
Heparin
Fig 1. Arm skin tempen#un' at tiw ~ite of the bleeding time determination before and 5 minutes ~f?er the administration off heparin in 55 study patienG. The means ~ stamtard error of the mean for eaci~ time point are shown.
aged 212,833 + 66,569//.tL. Five minutes after heparin administration it averaged 198,040 ÷ 56,451//~L (p - 0.24). Concentration
and Activated
Clotting Time
The activated clotting time (ACT) before heparin administration ranged from 105 to 201 seconds and averaged 137.9 +_ 19.2 seconds. In most of the patients, heparin was administered to achieve an ACT of more than 600 seconds. As a result, the ACT after the administration of the first dose of heparin ranged from 479 to 1,000 seconds and averaged 870 + 168 seconds. Heparin concentration in the blood before heparinization was 0.22 +_ 0.11 IU/mL. After the first dose, it ranged from 0.2 to 9.4 1U/mL and averaged 6.26 + 1.53 IU/mL. There was no correlation b et ween the ACT and the blood heparin concentration (r 0.09) after the administration of the first dose of heparin. Platelet Function
Platelet function parameters before and 5 minutes after heparin administration are shown in Table 1. The bleeding time before heparin administration ranged from 3.5 to 10.5 minutes and averaged 6.3 minutes. After the
Table 1. Platelet Function Parameters Bt~ti~re a m t 5 M i n u t e s A f t e r A d m i n i s t r a t i o n o f Heparin
Parameter
n
Before Heparin
After Heparin
p Value
Bleeding time (minutes) Shed blood T x B 2 (pg/0.1 ml) vWF antigen (%)
54
6.3 - 2.1
12.6 - 4.9
0.00001
538 - 187
0.00002
136 " 55
0.64
TxB 2 = thromboxane
32
1152
15 Bz;
-
669
172 r 48 vWF
= vml Willebrand
~-
Pre Heparin
Post
Pre
Heparin
"\\
I..- 10 o? c
factor.
.
o't
Post Heparin
,. Pre
Heparin
Post Heparin
[ i\, 2. Bleeding time and shed blood thromboxane B, in the patients in whom paired measurements were obtained before and 5 minutes after the administration of heparin. The means +_ standard error of the mean for each time point are shown.
administration of heparin, the bleeding time increased in all but 2 patients (Fig 2); it averaged 12.6 minutes. This was a marked and a significant increase c o m p a r e d with the p r e h e p a r i n level (p < 0.00001). The administration of heparin also resulted in a m a r k e d fall in the level of thromboxane B 2 in the blood shed from the site of the bleeding time determination. Paired data were obtained in 32 patients; their results are shown in Table 1 and Figure 2. The m e a n shed blood t h r o m b o x an e B2 level before heparin administration was 11.5 ng/mL. After heparin administration, it fell to 5.5 n g / m L (p - 0.00002). There was a moderate but significant negative correlation between the bleeding time and the shed blood thromboxane B2 (r = -0.43; p < 0.0005). There were no significant negative correlations b e t w e e n the skin t e m p e r a t u r e and the changes in the corrected bleeding time and the shed blood t h r o m b o x an e B2 level. Therefore, the changes in the bleeding time and t h r o m b o x an e B2 were not due to the slight fall in skin t e m p e r a t u r e that was observed between the preheparin and postheparin time points. Von Willebrand factor antigen concentration was measured before and 5 minutes after heparin administration in 15 patients. As shown in Table 1, the vWF antigen concentration before and after heparin were not significantly different. The vWF m u l t i m e r distribution was not altered in patients after infusion of heparin, as m e a s u r e d by visual inspection of vWF antigen on autoradiographs (Fig 3). Fibrinolytic Activity
Table 2 displays the results of fibrinolytic and related parameters before and after the administration of heparin. Plasmin was m e a s u r e d in 19 patients. As shown in Figure 4, heparin administration resulted in a tenfold increase in plasmin from a p r e h e p a r i n average of 11.8 U / L to a postheparin average of 125.4 U/L (p < 0.0001). In
Ann Thorac Surg 1995;60:1008 14
KHURI ET AL HEPARIN EFFECTSON PLATELETSAND FIBRINOLYSIS
200
n=16 p< 0001
1011
n=51 p=O.05 1500-
150
~
/
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t
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50 11/
250
0 A
N
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A
1
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2
A
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3
Pat i en t Fi,~,,3. Autoradio<\,raph of yon Wilh'br, nzd tactor in normal pool plasma (N), rout.from 3 retu'esclttativt' patients q/1,5,b
these patients, the preheparin and postheparin administration levels of plasmin correlated significantly with the preheparin and postheparin bleeding times (r 0.75; p 0.005) (Fig 5). In 51 patients in whom D-dimer measurements were made, the administration of heparin elicited an increase in D-dimer level in 43 (77%) and no change or a decrease in 12 (23",h) (see Fig 4). On the average, there was a modest but significant increase in the D-dimer level from a preheparin mean of 571.3 ~g/mL to a postheparin mean of 698.5/~g/mL (p 0.05). As shown in Table 2, the administration of heparin elicited no significant changes in the plasma levels of fibrinogen, tissue plasminogen activator, plasminogen, antiplasmin, and antithrombin It1.
Comment
Parameter
n 16 51 25 28
9.7 571.3 297.1 7.12 5.1 387.7 84. I 11,H
+
125.4 " 698.5 : 6.47 372.8
34.8 I' 358.6h 5.17I" 109.1l'
Posl Heparin
Plateh't D y s f u n c t i o n in O p e n Heart Operations and the Eft-cot o f H e p a r i n It has long been recognized that patients u n d e r g o i n g cardiac operations have increased postoperative bleeding. This has been attributed to a cardiopulmonary byp a s s - i n d u c e d hemostatic defect, which was thought to be
175
150 '
i I
Value O.OI)(ll 0.05
j
125 -i
I'
~100
Plasmin (U/L) D-dimer (/xglmL) t-PA (nglmL) Fibrinogen (mg/dL) b Plasminogen (",) Antiplasmin C,,) Antithrombin 111 (%,)
t
Pre Heparin
before the institution of cardiopulmonary bypass, causes platelet dysfunction (manifested by a prolongation of the bleeding time and a reduction in the ability of the platelet to produce thromboxane A z in vivo) and increased fibrinolvsis (manifested by increased plasma levels of plasmin and o-dimer). Hence, heparin contributes to the hemostatic defect observed in patients u n d e r g o i n g cardiac operations not only by inhibiting coagulation through its effect on a n t i t h r o m b i n III, but also by eliciting direct adverse effects on the platelet and the fibrinolytic system.
Lllllt" 2. Parameters Related to Fibrinolysis Before amt 3 Minutes Afitcr Administration of Hcparin
,After Heparin
%st Heparin
lik~4. Plasma h'vels of plasmin and u-dimer in the patients in whom paired measurements were obtained before and 5 minutes l{fter the administration qf heparin. The means - standard error of the mean for each time point are shown.
This study demonstrates that, in patients undergoing open heart operations, the administration of heparin,
Before 1teparin
o [
Pre Heparin
E
jJ~
-
"
I
75 -4
•
NS NS
50
NS XS NS
25
i
/O'"
,.'J
•
r = 0.75
p = 0,005
19 19 28
79.3 75.2 SO
I2.~q 8.4 l2
84 71 8(I
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7
9
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i
i
13
15
17
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21
Bleeding Time (minutes) "~ \"alue
NS
corrected
Inr d i ] u t h l n ,
I]Ot S i g l l i t i c a n l ;
l-P\
h Sdill[~lt'- n e u l r a l i / e d w i t h prlil,lnlint,. [iSSLIC t~l
Fi~,,5. Correlation between bleedit g tinw and plasma plasmin levels l,ctbrc and ,5 minutes after the administration of heparin.
1012
KHURI ET AL HEPARIN EFFECTS ON PLAH 1.111S ;\NI) FIBRINOI.YSIS
due mostly to platelet dysfunction and, to a lesser extent, increased fibrinolysis [1, 101. Because the magnitude of thrombocytopenia encountered in the course of a cardiac operation is not severe enough to account for the marked extension of the bleeding time observed during and after cardiopulmonaD ~ bypass [2], investigations in this field have focused on specific platelet abnormalities that might be induced by cardiopulmonary bypass. The platelet defect of cardiopulmonary bypass has been postulated to be due to a loss of the platelet membrane receptors responsible for platelet adhesion and aggregation. Loss of platelet m e m b r a n e receptors for both the vWF and fibrinogen were reported during and after cardiopulmonary bypass II1-13]. However, the methods with which the platelets were prepared in these studies involved centrifugation and gel filtration of the platelets before assay, thereby introducing the possibility of an artifactual in vitro decrease in platelet surface GPIb-IX complex (the vWF receptor) and GPllb-llla complex (the fibrinogen receptor) as a result of proteolysis or activation. In a study of 20 patients undergoing cardiac operations, we used a flow cytometric method that allowed us to study the platelet GP[b-IX and GPllb/llla in whole blood, thereby avoiding potential artefactual reductions in platelet surface glycoprotein receptors. With this method, we demonstrated that cardiopulmona D, bypass did not result in a decrease in the platelet surface expression of either the GPIb-IX complex or the GPllbIlia complex [3]. This study underscored the possibility of factors extrinsicto the platelet itself that might contribute to the platelet dysfunction observed in patients undergoing cardiac operations. Two such factors are hypothermia and heparin. In a study of 37 patients undergoing cardiopulmonary bypass, we confirmed the adverse effect of hypothermia on platelet function by demonstrating that hypothermia resulted in a significant prolongation of the bleeding time and a significant reduction in the ability of the platelet to produce thromboxane A 2 [4[. Platelet function in the present study was assessed bv the measurement of the bleeding time and the level of thromboxane B 2 in the blood shed from the skin at the site of the bleeding time determination. Despite its limitations, the bleeding time continues to be a reliable indicator of platelet function and platelet-vessel wall interaction [1]. The measurement of the shed blood thromboxane B 2 has also been shown to be a reliable indicator of platelet function [2, 14]. It has the distinct advantage of reflecting in vivo platelet function, making it an ideal measurement for use in patients undergoing cardiopulmonary bypass [2-4]. Both measurements indicated a marked reduction in platelet function after the administration of heparin. Although it has been long recognized that heparin caused a prolongation of the bleeding time [15], its recognition as an etiologic factor in postbypass hemorrhage is only recent [1, 161. The mechanism with which heparin might cause platelet dysfunction was partially addressed in our previous study in which whole blood flow cytometry was employed in the characterization of the platelet membrane glycoproteins Ib and IIb-llIa and platelet activation be-
Ann Thorac Surg 1995;60:1008-14
fore and after cardiopulmonary bypass [3]. That study provided evidence that heparin suppressed platelet activation in vivo via inhibition of endogenousthrombin, the latter being the most important platelet agonist in vivo. Another possible mechanism for the inhibition of platelet function by heparin is through an inhibition of vWF activity. Ristocetin is a cationic antibiotic that induces in vitro binding of vWF to its receptor on platelet GPlb. Sobel and associates [17] demonstrated that the intravenous administration of heparin to patients before open heart operations reduced ristocetin cofactor activity by 58%. This impairment of vWF-dependent platelet function was closely related to plasma heparin levels but not to plasma vWF levels. It appeared to be caused by direct binding of heparin to vWF in solution, but may also be a consequence of heparin binding directly to the platelet surface [18]. In the present study, we have demonstrated that the concentration and multimer distribution of the vWF were unaffected by the administration of heparin. Hence our data are consistent with the hypothesis that heparin reduces platelet function by preventing the vWF from binding to its receptor on the GPlb on the platelet. The third mechanism by which heparin can induce platelet dysfunction is through its effect on plasmin. This is explained in the section below.
Fibrinolysis During Cardiopulmonary Bypass and the Effect of Heparin There are a number of pathways that could lead to increased fibrinolysis during cardiopulmonary bypass operations, including the activation of kallikrein and the release of tissue plasminogen activator from the endothelial cell [1[. Fibrin(ogen) degradation products and D-dimer levels increase during and following cardiopulmonary bypass ]1, 19]. Administration of aprotinin during cardiopulmonary bypass prevents the formation of fibrin degradation products and the reduction in %antiplasmin activity [20]. It also reduces the bleeding time ]21[. These observations, the mechanism of action of aprotinin [22], and the clinical effectiveness of aprotinin in decreasing blood loss ]21[, strongly suggest that fibrinolysis is important in the hemorrhagic diathesis associated with cardiopulmonary bypass operations. The present study demonstrates that, in patients before the institution qf cardiopulmona~ bypass, the administration of heparin increases fibrinolysis as evidenced by a uniform increase in plasmin and a partial but significant increase in D-dimer level. Heparin and its fractions, however, have been known to possess profibrinolytic activities for more than a decade [5]. Fareed and associates [5] reported an increase in tissue plasminogen activator levels in h u m a n volunteers after the institution of intravenous and subcutaneous heparin over a period of 10 days (daily dose of 7,500 units). They postulated a n u m b e r of profibrinolytic actions of heparin and its fractions. Several recent studies have shown that heparin and heparin-like compounds stimulated cell surface plasminogen activation by 10- to 17-fold [23]. Because this interaction occurred at the cell surface level, it would be unlikely that
Ann Thorac Surg 1995;60:1{)1)8 14
the profibrinolytic effect of heparin would be reflected bv the levels of plasminogen in the plasma. Hence, in our study, we did not show anv change in the plasma levels of plasminogen with the administration of heparin. The lack of a significant increase in tissue plasminogen activator after the administration of heparin did not denote a lack of profibrinolytic activity because the plasma level tissue plasminogen activator antigen may not correlate with the functional activity of the molecule in patients u n d e r g o i n g cardiopulmonary bypass. The rise in plasmin and o-dimer levels after the administration of heparin indicated an increase in profibrinolytic activity. It is of note that plasmin, fibrin degradation products, and Ddimer have all been shown to interfere with platelet aggregation, p r e s u m a b l y through a proteolytic effect on the platelet GPI|Ia in the case of plasmin [24] and by competing with fibrinogen for GPIIb/llIa b i n d i n g in the case of the degradation products. Hence the generation of plasmin, stimulated bv heparin, provides another possible mechanism by which heparin impairs platelet function as well. A confirmatory finding of this mechanism is the significant correlation that was observed in this study between the bleeding time and the plasma levels of plasmin.
Clinical Implications The hemostatic defect observed in patients undergoing cardiac operations is not fullv reversed with the administration of protamine and the discontinuation of cardiop u l m o n a r y bypass. In fact, D-dimer levels increase after the administration of protamine, and the bleeding time continues to be significantly elevated in the hours after the discontinuation of cardiopulmona D, bypass [1, 2]. The bleeding time at 2 hours after cardiopulmona D' bypass was shown to be an important d e t e r m i n a n t of postoperative blood loss [2]. Interventions aimed at reducing postoperative platelet dvsfunction and fibrinolysis are likely to reduce postoperative blood loss in patients u n d e r g o i n g cardiac operations. Because a variety of complex factors could contribute to the hemostatic defect observed in the initial postoperative hours in these patients, it is very difficult to appreciate the specific contribution of the administration of heparin per se to this defect. Nevertheless, the present study raises questions as to whether an alternative anticoagulant or a lower dose of heparin might improve the hemostatic balance during open heart operations. In the present study, the ACT was kept on the high side to achieve full suppression of thrombin. Significantly lower ACT levels have been employed in conjunction with heparin-coated surfaces. It remains to be determined whether combining low-dose heparin with heparin-coated surfaces would suppress thrombin adequately and, at the same time, reduce the adverse effect of heparin on platelet function a n d fibrinolysis. It also r e m a i n s to be d e t e r m i n e d whether an alternative anticoagulant that would not interfere with either the platelet function or the fibrinolytic process can improve postoperative hemostasis in the cardiac surgical patient.
KHURIET AL HFPARIN EFFF,CTS ON PLATELETSAND FIBRtNOLYSIS
1013
We acknowledge the valuable help of Jennifer Marjani and Frank Glavin in the preparation of the manuscript. Supported by the US Navy (Office of Naval Research Contract N00014-94-C-0149 with funds provided by the Naval Medical Research and Development Command), and by the Richard Warren Surgical Research and Educational Fund, Westwood, Massachusetts.
References I. Khuri SF, Michelson AD, Valeri CR. The effect of cardiopulmonary bypass on hemostasis and coagulation. In: Loscalzo J, Schafer AI, eds. Thrombosis and hemorrhage. Cambridge, MA: Blackwell Scientific, 1994:1051-73. 2. Khuri SF, Wolfe JA, Josa M, et al. Hematologic changes during and after cardiopulmonary bypass and their relationship to the bleeding time and nonsurgical blood loss. J Thorac Cardiovasc Surg 1992;104:94-107. 3. Kestin AS, Valeri CR, Khuri SF, et al. The platelet function defect of cardiopulmonary bypass. Blood 1993;82:107-17. 4. Valeri CR, Khabbaz K, Khuri SF, et al. Effect of skin temperature on platelet function in patients undergoing extracorporeal bypass. J Thorac Cardiovasc Surg 1992;104:108-16. 5. Fareed J, Walenga JM, Hoppensteadt DA, Messmore HL. Studies on the profibrinolytic actions of heparin and its fractions. Semin Thromb Hemost 1985;11:199-207. 6. Pennv W, Weinstein MJ, Ware JA, Salzman E. Correlation of circuiating yon Willebrand factor levels with cardiovascular henlodynamics. Circulation 1991;83:1630-6. 7. Weinstein MJ, Ware JA, Troll J, Salzrnan EW. Changes in yon Willebrand factor during cardiac surgery: effect of desmopressin acetate. Blood 1988;71:1648-55. 8. Babson SR, Babson AL. Development and evaluation of a disposable device for performing simultaneous duplicate bleeding time determinations. Am J Clin Pathol 1978;70: 406- 8. 9. Valeri CR, MacGregor H, Pompei F, Khuri SF. Acquired abnormalities of platelet function. [Letter]. N Engl J Med 1991;324:1670. 10. Woodman RC, Harker LA. Bleeding complications associated with cardiopulmonary bypass. Blood 1990;76:1680-97. II. Rinder CS, Mathew JP, Rinder HM, Bonan J, Ault KA, Smith BR. Modulation of platelet surface adhesion receptors during cardiopulmonary bypass. Anesthesiology 1991;75:563-70. 12. Van Oeveren W, Harder MP, Roozendaal KJ, Eijsman L, Wildevuur CRH. Aprotinin protects platelets against the initial effect of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99:788-96. 13. Wenger RK, Lukasiewicz H, Mikuta BS, Niewiarowski S, Edmunds LH. Loss of platelet fibrinogen receptors during clinical cardiopulmonary bypass. J Thorac Cardiovasc Surg 1989;97:235-9. 14. Gerrard JM, Tavback S, Singhroy S, et al. In vivo measurement {}f thromboxane B2 and 6-keto-prostaglandin F1.~ in humans in response to a standardized vascular injury and the influence of aspirin. Circulation 1989;79:29-38. 15. Heiden D, Mielke CH, Rodvien R. Impairment by heparin of primary haemostasis and platelet (C14) 5-hydroxytryptamine release. Br J Haematol 1977;36:427-36. 16. John LCH, Rees GM, Kovacs lB. Inhibition of platelet function b~ heparin. J Thorac Cardiovasc Surg 1993;105:816-22. 17. Sobel M, McNeill PM, Carlson PL, et al. Heparin inhibition of yon Willebrand factor-dependent platelet function in vitro and in vivo. J Clin Invest 1991;87:1787-93. 18. Horne MK Ill, Chao ES. Heparin binding to resting and activated platelets. Blood 1989;74:238-43. 19. Giuliani R, Szwarcer E, Martinez Aquino E, Palumbo G. Fibrin-dependent fibrinolytic activity during extracorporeal circulation. Thromb Res 1991;61:369-73. 2(}. Blauhut B, Gross C, Necek S, Doran JE, Spath P, Lundsgaard-]tansen P. Effects of high-dose aprotinin on blood
1014
KHURI E~f AL HEPARIN EFFECTS ON PLATEI]{IS ANI) FIBRINOLYSIS
loss, platelet function, fibrinolysis, c o m p l e m e n t , a n d renal function after c a r d i o p u l m o n a r y b y p a s s . J T h o r a c C a r d i o v a s c S u r g 1991;101:958- 67. 21. B i d s t r u p BP, Royston D, Sapsford RN, 1-aylor KM. R e d u c t i o n in blood loss a n d blood u s e after c a r d i o p u l m o n a r y b y p a s s with h i g h d o s e aprotinin. J Thorac C a r d i o v a s c S u r g 1989;97: 364-72. 22. M a n n u c c i PM. N o n t r a n s f u s i o n a l modalities. In: Loscalzo ],
Ann Thorac Surg 1995;60:1008-14
Schafer AI, eds. T h r o m b o s i s a n d h e m o r r h a g e . C a m b r i d g e , MA: Blackwell Scientific, 1994:1117-35. 23. Rijken DC, de M u n k GA, Jie AF. i n t e r a c t i o n s of p l a s m i n o g e n activators a n d p l a s m i n o g e n with heparin: effect of ionic s t r e n g t h . T h r o m b H a e m o s t 1993;70:867-72. 24. O u i m e t H, Loscalzo J. Reciprocating autocatalytic interactions b e t w e e n platelets a n d the p l a s m i n o g e n activation s y s t e m . T h r o m b Res (in press).
DISCUSSION D R REPHAEL M O H R ( T e I - H a s h o m e r , Israel}: In our experience we e v a l u a t e d t h e effect of v a r i o u s s t a g e s of p r e b y p a s s h e p a r i n a d m i n i s t r a t i o n a n d so on on platelet function with v a r i o u s m e t h o d s , i n c l u d i n g in vitro, s t a n d a r d PLP aggregation, a n d w h o l e blood a g g r e g a t i o n , a n d we also e v a l u a t e d it on the r e s p o n s e of the blood from t h e patients to the extracellular matrix m o d e l as v i e w e d by electron microscopy. In all t h o s e s t u d i e s t h e initial plalelet d y s f u n c t i o n o b s e r v e d ~ as always after initiation of c a r d i o p u l m o n a D, b y p a s s a n d the levels of d y s f u n c tion were a l m o s t always o b s e r v e d w h e n b y p a s s t e m p e r a t u r e w e n t down. However, w h e n e v e r we m e a s u r e d the effect of h e p a r i n a d m i n i s t r a t i o n on platelet ftmction with all t h e s e rnodels, we n e v e r were able to d e m o n s t r a t e w h a t yotl just s h o w e d us, that platelet function is in a n y w a v d e r a n g e d by h e p a r i n a d m i n istration. How can w~u explain this discrepancy? D R KHURI: O t h e r investigators, e m p l o y i n g various m e t h o d o l ogies, h a v e also s h o w n recently that h e p a r i n c a u s e s significant platelet dysfunction. In this s t u d y we have u s e d a very reliable m e a s u r e of in vivo platelet function, w h i c h is the ability of the platelet to p r o d u c e t h r o m b o x a n e m r e s p o n s e to a b l e e d i n g insult. I can only explain the d i s c r e p a n c y b e t w e e n o u r findings a n d y o u r s on the basis of possible m e t h o d o l o g i c differences. D R S A F U H A T T A R {Baltimore, MD): 1 c o n g r a t u l a t e Dr Khuri a n d his associates on a w e l l - d e s i g n e d study. In the early 1970s 1 p r e s e n t e d a p a p e r on the effects of h e p a r i n on platelets at the IVth International C o n g r e s s on T h r o m b o s i s a n d H e m o s t a s i s . A g g r e g a t i o n w a s i n d u c e d by h e p a r i n tested in platelet-rich p l a s m a . The effect of h e p a r i n on platelet a g g r e g a tion was f o u n d to be proportional to the c o n c e n t r a t i o n ot h e p a r i n . We also c o m p a r e d the effect of intestinal h e p a r i n v e r s u s l u n g heparin; there w a s a significant increase in the a g g r e g a t i o n of the platelets w h e n intestinal h e p a r i n w a s u s e d in c o m p a r i s o n with t h e l u n g heparin. T h e r e a s o n is that h e p a r i n is a h e t e r o g e n e o u s s u b s t a n c e that p r o b a b l y has bits of intestines, spleen, a n d o t h e r t h i n g s in it; therefore, t h e s e t i s s u e s are the o n e s that p r o d u c e the platelet aggregation. My q u e s t i o n s to Dr Khuri are the following. First, did vou u s e t h e platelet c o u n t as a function? T h e c o u n t of the platelets does not m e a n very m u c h ; the platelets m a v be f u n c t i o n l e s s a n d vet t h e c o u n t m a y be all right. Second, w h a t kind of h e p a r i n did you use, intestinal or lung h e p a r i n ? Third, if you use a m o r e purified h e p a r i n , will it m i n i m i z e the effects of h e p a r i n on the platelets? D R L. HENRY E D M U N D S (Philadelphia, PA): Doctor Khuri, 1 e n j o y e d y o u r p a p e r a n d l enjoyed the abstract, a n d 1 a m familiar with y o u r work with Drs Kestin a n d Mickelson. [ t e p a r i n is a w e l l - k n o w n a g o n i s t for platelets, b u t also it is a less well-kno~x n a g o n i s t for n e u t r o p h i l s a n d c o m p l e m e n t . It is certainly not an ideal a n t i c o a g u l a n t by a long shot. O u r g r o u p believes that t h e r e is an e,xtrinsi¢ factor that s u p p r e s s e s platelet function, as w m r g r o u p h a s p o i n t e d out. However, I have to ask you w h e t h e r you
think t h e r e is an intrinsic defect p r o d u c e d by c a r d i o p u l m o n a r y b y p a s s in platelets, as o u r data lead u s to believe. I a m d i s t u r b e d by y o u r c o n c l u d i n g c o m m e n t , that m a y b e a c o m b i n a t i o n of s u r f a c e - b o u n d h e p a r i n a n d s o m e o t h e r anticoa g u l a n t will get us a r o u n d this p r o b l e m . R e c o m b i n a n t h i r u d i n is the alternative a n t i c o a g u l a n t that is b e i n g c o n s i d e r e d the most. This is a v e D' t i g h t - b i n d i n g inhibitor of t h r o m b i n , b u t it does n o t p r e v e n t the h~rmation of t h r o m b i n . It is i m p o r t a n t for e v e r y o n e w h o does cardiac s u r g e r y to realize that t h r o m b i n is f o r m e d in progressively i n c r e a s i n g a m o u n t s d u r i n g every, c a r d i o p u l m o narv b y p a s s operation, a n d that it is a d a n g e r o u s e n z y m e to circulate. So the idea of u s i n g s u r f a c e - b o u n d h e p a r i n with a direct t h r o m b i n inhibitor s u c h as h i r u d i n h a s little a p p e a l to m e . S u r f a c e - b o u n d h e p a r i n d o e s n o t a p p e a r to h a v e an a n t i c o a g u lant effect a n d therefore is unlikely to help. W o u l d y o u c o m m e n t on w h e t h e r you believe, or y o u r g r o u p believes, that t h e r e is an intrinsic defect in platelets p r o d u c e d by cardiopulmonary bypass? DR KHURI: T h a n k you, Dr E d m u n d s . 1 a m very appreciative of v~mr c o m m e n t s . I can very well u n d e r s t a n d y o u r c o n c e r n a b o u t m y r e m a r k r e g a r d i n g the possible a d v a n t a g e of c o m b i n i n g low-dose h e p a r i n with h e p a r i n - c o a t e d circuits, a n d 1 look forward to the publication of y o u r data which, as I i n t e r p r e t f r o m w m r c o m m e n t s , will s h o w that s u c h a c o m b i n a t i o n w o u l d n o t a d e q u a t e l y p r e v e n t the f o r m a t i o n of t h r o m b i n , which, 1 agree, is a molecule we n e e d to avoid d u r i n g b y p a s s . In the p r e s e n t study, we m a i n t a i n e d relatively h i g h activated clotting time levels (an a v e r a g e of 700 to 800 s e c o n d s ) to e n s u r e specifically that t h e t h r o m b i n was a d e q u a t e l y neutralized. As to the q u e s t i o n of w h e t h e r the c a r d i o p u l m o n a r y b y p a s s i n d u c e d platelet defect is intrinsic or extrinsic to the platelet, o u r s t u d i e s to date h a v e identified h y p o t h e r m i a a n d h e p a r i n as extrinsic factors r e s p o n s i b l e , in part, for this platelet d y s f u n c tion. O u r s t u d i e s h a v e not r u l e d o u t the p r e s e n c e of a cardiop u l m o n a r y b y p a s s - i n d u c e d intrinsic platelet defect. As m y coll e a g u e s a n d 1 have p u b l i s h e d in Blood, however, the m e c h a n i s m of this intrinsic defect is not likely to be a loss of t h e platelet m e m b r a n e receptors GPIb a n d GPlIb-IIla, as earlier s t u d i e s h a d suggested. Doctor Attar, we appreciate the fact that y o u were w a y a h e a d of your time in s h o w i n g , m o r e t h a n 20 y e a r s ago, that h e p a r i n c a u s e d a b n o r m a l platelet aggregation. W e h a v e not u s e d platelet a g g r e g a t i o n in the a s s e s s m e n t of platelet function b e c a u s e of the variability in that t e c h n i q u e . Instead, we h a v e o p t e d for t h e m viw~ test of m e a s u r i n g t h r o m b o x a n e B2 in t h e blood s h e d at the site of the b l e e d i n g time d e t e r m i n a t i o n . W e believe t h a t this test of platelet f u n c t i o n is m o r e applicable a n d r e l e v a n t to t h e patient u n d e r g o i n g c a r d i o p u h n o n a r y b y p a s s . M a y b e that is w h y t h e r e is a d i s c r e p a n c y b e t w e e n o u r data a n d t h o s e of Dr M o h r from Israel. As to y o u r specific q u e s t i o n s a b o u t h e p a r i n , we h a v e u s e d porcine h e p a r i n , a n d we have h a d no experience with t h e m o r e purified f o r m s of heparin.