Heparin-induced platelet aggregation in burn patients

THROMBOSIS

RESEARCH

Volume1, Pages291-299,PergamonPress,1977.Printedin Gt. Britain.

HEPARIN-INDUCEDPLATELET AGGREGATIONIN BURN PATIENTS

J. Allen Mims, Kay E. Sarji, James S. Kleinfelder, and Karl Eurenius Department of Medicine, Medical Universityof,South Carolina and Veterans AdministrationHospital, Charleston,South Carolina 29401.

(Received 4.6.1976; in revised form 23.12.1976. Accepted by Editor K.M. Brinkhous)

ABSTRACT Platelets from patients with thermal injury were studied to evaluate the effect of beef lung and intestinalmucosal heparin upon platelet aggregation. In contrast to controls, 15% of samples from burn patients demonstratedspontaneous aggregation,and 60% showed either first or second phase aggregation after exposure to either heparin preparation. No differencewas observed between burn patient and normalcontrol platelet@in their aggregationresponse to ADP. Increased spontaneousand heparin-inducedaggregationseen in burn patient platelet-richplasma could not be correlatedwith platelet count, sampling time, or plasma factors. The enhanced response to heparin seen in these patients appears to be intrinsic to the platelet.

INTRODUCTION Although heparin is widely used as an anticoagulant,it appears also to have some procoagulantactivity. This paradox was first suggestedby the appearance of thrombocytopeniafollowing the initiationof heparin therapy (1, 293). Disseminatedintravascularcoagulationand heparin-inducedplatelet aggregationhave both been demonstratedin,patientssubsequentto heparin therapy (4,5). Because these findings suggest that heparin may be contraindicated in disorderswhere platelet hyperfunctionis critical,and since stress has been shown to enhance platelet aggregation (6), we sought to evaluate heparin's in vitro effects on platelets obtained from patients who have undergone a major stressful event: i.e., thermal injury. Our study reveals that burn patients may have both hyperaggregableplatelets and spontaneousaggation as well as a 30% incidenceof second phase heparin-inducedaggregation.

291

292

HEPARIN

AND PLATELET

MATERIALS

AGGREGATION

Vol.lO,No.2

AND METtlOL)S

Platelet Collection: Thirty-fourblood samples were collected from 7 patients with thermal burns ranging from 8.5 to 90% total body surface. Samples were collected upon arrival of the patient at the Bum Unit of the Medical Universityof South Carolina and on a weekly basis thereafter. Ten normal volunteerswho had not taken medication for 10 days were used as controls. Blood was collected in 3.8% trisodium citrate (1:9 V/V) and platelet-richplasma (PRP) was prepared by centrifugationat 100 x g for 10 minutes at room temperature. Plateletpoor plasma (PPP) was prepared by centrifugationat 2000 x g for 10 minutes and samples were frozen for later studies. Platelet-richplasma was kept at room temperatureand aggregationstudies were completedwithin three hours of collectiontime. All manipulationswere performed using plastic ware. Reagents: Adenosine diphosphate (ADP) (Sigma Chemical Co., St. Louis, MO) was diluted to working concentrations,0.8 uM, with Verona1 buffered saline, pH 7.40. This concentrationwas selected since this dose produced minimal but definite second phase platelet aggregation. Bovine lung and intestinalmucosal heparin (Upjohn Co., Kalamazoo,MI), 10,000 U/ml, were diluted to working concentrationswith 0.9% sodium chloride. Platelet Aggregation: Platelet counts were performed using phase microscopy after dilutionwith 1% ammonium oxalate. Platelet studies were performed using a platelet aggregometer (Chronolog)at 370C and stirrine at 1200 rum with a horizontal stirring bar. PRP, 0.45 ml, was initially stirred in the aggregometerto test for spontaneousaggregation. At two minutes, 0.05 ml saline or lung or intestinal mucosal heparin was added to the cuvette. If spontaneousor heparin-induced aggregationdid not occur within five minutes, 0.8 uM AUP was added to the mixture and recordingwas continued for four minutes. To determine approximate area under the curve, recordingswere traced, the area from initial to maximal aggregation cut out and weighed with a laboratorybalance. RESULTS

Figure 1 indicates that 3.6 U/ml of beef lung heparin produced maximal enhancementof ADP-inducedplatelet aggregationas measured by assessing the area under a four minute aggregationcurve. For most studies, this concentration of heparin was used. Five of 24 burn samples demonstratedspontaneousaggregationprior to the addition of heparin or ADP as indicated in Table 1. Additionally,over 60% of burn patient plasma samples aggregated after heparin addition and more than half of these demonstratedsecond phase aggregation. All 34 samples of burn patient PRP were exposed to either beef lung and/or intestinalmucosal heparin. Fourteen of these samples demonstratedsecond phase aggregationduring the five minute heparin incubationperiod, and 13 showed first phase aggregation as indicatedby a slow but definite increase in baseline optical density. This latter aggregationwas confirmedby phase microscopy,and examples of both fast and slow heparin-inducedaggregationare compared to a saline control in Figure 2.

Vol.lO,No.2

HEPAR

AND PLATELET AGGREGATION

2 H6parin

4

6

293

16

Concentmtion W/mlPRP)

FIG. 1 Percent increase in ADP-induced platelet aggregation of normal platelet-rich plasma as a function of beef lung heparin concentration. Aggregation in these studies was measured as the area under the four minute aggregarion curve, where x = mean change in aggregation for each of the four experiments and dark circles equal mean values for all experiments. PRP concentrations were adjusted to 400,000 platelets/mm3. Figure 3 indicates the enhancement of aggregation when spontaneously aggregating PRP was incubated with either intestinal mucosal or beef lung heparin. The five such samples encountered among three different patients all demonstrated this heparin sensitivity even in their already hyperaggregable state. Table 2 indicates the change in ADP-induced platelet aggregation when burn patient PRP was preincubated with either beef lung or intestinal mucosal heparin. Prior exposure to either heparin preparation produced enhancement of ADP-induced aggregation in both normal and burn PRP. There appeared to be no difference in this enhancement in both platelet populations, and enhancement was the same with each heparin preparation. Area and optical density enhancement of first phase aggregation were approximately 1.5 x control and of second phase aggregation, approximately 2.5 x control. Less enhancement was noted in measurements of aggregation slope. Figure 4 demonstrates the enhancement of ADP-induced aggregation by both intestinal mucosal and beef lung heparin in one patient sample. Since most of our patients were referred to this bum center at variable periods postbum, the information was calculated regarding mean day of collection (+ SEM) and mean platelet-rich plasma concentration (PRP x 10m3 + SEM). Among the bum patients, five spontaneous aggregation samples had PRP counts of 282 + 62 and collection day range of 43 + 23; 12 heparin-induced aggregation samples had counts of 270 + 58 and a range of 27 + 10 days; 15 samples falling in neither of these two categories had counts of 239 f 25 and a range of 21 + 6 days. Ten control samples had PRP counts of 292 + 45. It appeared

HEPARIN AND PLATELET AGGREGATION

294

TOTAL

SANPLES

34 SrnLeS

TOTAL PATIENTS

9 PATIEWTS

1

sFoNTANEQus AamlNwIoN

NZPMIN

INDUCED ACCNEGATIGI

SCKF Lmlc

6129 FAST 0129 SUJH

INTESTINAL NUWSA

6115 FAST 5/15

WANIN

mINANcMENT

Vol.lO,No.2

SW

5 smLes

16129SMPLSS 11115SAWLSS

OF ADP

AOONECATION

SEEP LUNG

INTESTINAL NUWSA

21124DEFINITE 2124sLIcHT 9110DEPINIiK l/10SLIGHT

23121SAllsLSS

loll0su(pLSs

Incidence of spontaneous, heparin-induced, and heparinenhanced ADP platelet aggregation seen in 34 samples from nine different burn patients.

FIG. 2 Platelet aggregation as measured by optical density, as a function of time (in minutes) after addition of either heparin (3.6 U/ml) or saline to three separate plasma samples, indicating typical curves of "fast" and "slow" aggregation. ADP (0.8 pM) was added to the lower two samples at a later time in the study. that the average day of collection and PRP counts were not significantly different between control samples and bum patients demonstrating either spontaneous, heparin-induced, or only ADP-induced aggregation. To determine whether the increased sensitivity of platelets in burn PKP was due to plasma factors, to the platelet per se, or to a combination, the ability of various forms of platelet-poor plasma to enhance platelet aggregatic was determined. In these tests, the method of Kwaan et al (8) was used, in

Vol.lO,No.2

HEPARIN AND PLATELET AGGREGATION

295

FIG. 3 Platelet aggregationas measured by optical density is shown for one patient (48-6)who repeatedlydemonstratedspontaneous aggregation. Shown also in this panel are the aggregation curves of these same platelets when preincubatedat one minute with either beef lung or intestinalmucosal heparin.

TABLE 2 Percent change in AUP-inducedplatelet aggregationwhen all burn patient PRP were incubatedwith beef lung or intestinal mucosal heparin. Area of aggregationwas calculatedby weighing the area of paper falling either under first phase or four minute aggregation. Optical density changes were calculated by comparing the differencein optical density changes during the first phase or four minute aggregationin each sample. Slope of aggregationwas calculatedas the initial slope of first phase changes. In all studies; heparin concentrations were 3.6 U/ml and AUP concentrationswere 0.8 PM. Comparativestudies always utilized constant PRP concentrations and constant aggregometersettings. which 0.05 ml of test or control PPP was added to 0.4 ml of normal PRP in the aggregometerbefore the addition of ADP at a concentrationslightly below that necessary to produce second phase aggregation. Optical density differenceat four minutes after ADP addition was used as a measure of the ability of plasma to enhance aggregation. PPP from spontaneousaggregators(five samples), from heparin aggregators (five samples), and from five patients who demonstrated neither, were tested. Five PPP samples demonstratedenhancing activity. Three of these samples came from the first group and one from each of the latter two. It is of interest, that three of these five plasmas were from a single patient

296

at different ing activity

HEPARIN AND PLATELET AGGREGATION

times postburn. There was no correlation and either spontaneous or heparin-induced

Vol.lO,No.2

between plasma enhancaggregation.

Time in Minutes

FIG. 4 ADP-induced aggregation (0.8 pM) seen for one patient (48-l) when these samples were preincubated with either intestinal mucosal or beef lung heparin (3.6 U/ml PRP) or normal saline, indicating the typical enhancement of aggregation by heparin. DISCUSSION Thrombocytopenia is an early manifestation of burn injury (9,lO) and has been demonstrated in the scald burn rat model to be a reflection of sequestration of these cells by the burn wound (11). In this same animal model, we have previously demonstrated decreased platelet aggregation during this early period, related to the presence of a serum inhibitor (7). Later in convalescence, thrombocytosis has been documented both in burn patients (12) and in scald burn rats (ll), and is associated with enhanced ADP-induced platelet aggregation. Because patients with thermal injury frequently demonstrate clotting disorders which might require heparin (13,14,15), we elected to examine the influence of heparin on platelet aggregation in burn patients. Our findings indicate a significant incidence of spontaneous aggregation and a greater than 30% incidence of second phase heparin-induced aggregation. Furthermore, this enhancement in platelet function appears to occur independent of plasma factors in 3.8% titrated, non-attenuated platelet-rich plasma. The occasional ability of some PPP to enhance platelet aggregation is independent of the hyperaggreaggregation enhancgation seen in burn patient PRP. The presence of platelet ing factors in some plasmas may be related to undiagnosed diabetes, since this even in mild or latent diabetes (16). activity has been shown to be present The phenomenon in thermal injury appears intrinsic to the platelets. in vitro platelet aggregation is critically dependent upon laboratory exFriedlander (17), for example, reported an 8% incidence perimental conditions. of spontaneous aggregation in normal individuals when using a vertical stirwas related to the spering bar at a frequency of 4000 rpm. This observation Vreeken and van Aken (18) observed no spontancific conditions of stirring. using a horizontal magnetic stirrer eous aggregation in a normal population, Under these same conditions, 16 normal samples at a speed of 1200 rpm or less.

Vol.lO,No.2

HEPARIN

AND PLATELET

AGGREGATION

297

in our laboratory demonstrated no spontaneous aggregation. Spontaneous aggregation measured by numerous techniques has been observed in isolated, asymptomatic individuals (19), and in some patients with vascular disease (20). The effect of heparin on platelet function is even less clear. Numerous observations of thrombocytopenia occurring during heparin administration have been made (1,2,3). Rare observations of disseminated intravascular coagulation with heparin therapy also exist (4). Recently, Fratantoni (5) observed heparin-induced thrombocytopenia in a patient being treated for thrombophlebitis, and demonstrated heparin-induced aggregation in this patient's PRP after recovery. In washed platelets studied at 4OC, Eika (21,22) has observed heparin-induced platelet aggregation and has demonstrated a variation in this effect among various commercial preparations (23). Low temperature dependence in this system has been confirmed by Han and Ardlie (24) and Teien (25). Heparin-induced aggregation has also been demonstrated at 370C in unwashed platelets but appears to be limited to a first phase reaction (26). In our studies, we were unable to demonstrate significant second phase heparin-induced aggregation in any of our control PRP. Enhancement of ADP-induced platelet aggregation was seen to an equal degree in both our population and normal controls. This observation appears to be less unique and has been observed by others (27,28). No qualitative or quantitative differences were observed between each ui the two heparin preparations (beef lung, intestinal mucosal) in their ability to induce aggregation or enhance spontaneous or ADP-induced aggregation. Variations in platelet response toheparinshave been reported by others (23). The finding in our studies that platelets from burn patients appear both to aggregate spontaneously and to respond in a similar manner to therapeutic concentrations of heparin is, we believe, a significant observation. Hyperaggregability of platelets has recently been demonstrated by Yamazaki (29) in Patients with thromboembolic disorders. Thirty-one patients in acute stage of thrombosis were shown to have an enhancement of ADP and epinephrine-induced platelet aggregation. If hyperaggregability of platelets is causal and not a result of such thromboembolic disorders, then the observation of enhanced aggregation in our patient population may have clinical significance. Furthermore, since bum patients demonstrate thromboembolic phenomenon and are frequently treated with heparin anticoagulation, the observation of enhanced activity to heparin may also have important clinical elements. ACKNOWLEDGEMENT This work was supported by funds from the Upjohn Co., Kalamazoo, MI. REFERENCES 1.

NATELSON, E.A., LYNCH, E.C., ALFREY, C.P. JR. and CROSS, J.B. induced thrombocytopenia. Ann. Int. Med. 71, 1121, 1969.

2.

RHODES, G.R., DIXON, R.H. and SILVER, D. Heparin induced thrombocytopenia with thrombotic and hemorrhagic manifestation. Surg. Gynecol. Obstet. 136, 409, 1973,

3.

FIDLAR, E. and JAQHES, L.B. The effect of commercial heparin on the platelet count. J. Lab. Clin. Med. 33, 1410, 1948.

Heparin-

HEPARIN AND PLATELET AGGREGATION

298

Vol.lO,No.2

4. KLEIN, H.G. and BELL,W.R. Disseminated intravascular coagulation during heparin therapy.

Ann. Int. Med. 80, 466, 1974.

5. FRATANTONI, J.C., POLLET, R. and GRALNICK, HR. cytopenia: 395, 1975.

Heparin-induced thromboConfirmation of diagnosis with in vitro methods. Blood 45,

6. RRES, B.W.G. Technique for the estimation of abnormal disaggregation patterns in platelets.

Med. Lab. Technol. 32, 99, 1975.

7. EURENIUS, K. and RCTHBNBERG J.

Platelet aggregation after thermal injury. J. Lab. Clin. Med. 83, 355, 1974.

8. KWAAN, H.C., COLWELL, J.A., CRUZ, S. and SUWANWELA, N. let aggregation in diabetes mellitus. (Abstr). 9.

Increased plateJ. Clin. Invest. 50, 57a, 1971

CURRERI, P.W., KATZ, A.V., DOTIN, L.N., et al. Coagulation abnormalities in the thermally injured patient. Curr. Topics Surg. Res. 2, 401, 1970.

10.

GEHRKE, C.F., PENNER, J.A., NEIDERHUBER, J., et al. Coagulation defects in burned patients. Surg. Gynecol. Obstet. 133, 613, 1971.

11.

EURENIUS, K., MORTENSEN, R.F., MESEROL, P.M. and CURRERI, P.W. Platelet and megakaryocyte kinetics following thermal injury. J. Lab. Clin. Med. 79, 247, 1972.

12. EURENIUS, K., ROSSI, T.D., McEUEN, D.D., ARNOLD, J. and M&ANUS, Blood coagulation in burn injury. 1974.

W.F. Proc. Sot. Exp. Biol. Med. 147, 878,

13. McMANUS, W.F., EURENIUS, K. and PRUITI, B.A. JR. cular coagulation in burned patients.

Disseminated intravasJ. Trauma 13, 416, 1972.

14.

MCKAY, D.G. Trauma and disseminated intravascular coagulation. Trauma 9, 646, 1969.

J.

15.

ROBB, H.J. Dynamics of the microcirculation during a bum. 94, 776, 1967.

16.

COLWELL, J.A., SAGEL, J., CROOK, L., CHAMBERS, A. and LAIMANS, M. Correlation of platelet aggregation, "plasma factor“ activity and megathrombocytes in diabetic subjects with and without vascular disease. Metabolism, in press, 1976.

17.

FRIEDLANDER, I., COOK, I.J.Y., HAWKEY, C. and SYMONS, C. A laboratory study of spontaneous platelet aggregation. J. Clin. Pathol. 24, 232, 1971.

18.

VREEKEN, J. and van AKEN, W.G. Spontaneous aggregation of blood platelets as a cause of idiopathic thrombosis and recurrent painful toes and fingers. Lancet 2, 1394, 1971.

19.

KARDINAL, C.G., WEGENER, L.T. and ANDERSON, L.K. Spontaneous platelet aggregation occurrence in an asymptomatic individual. Am. J. Clin. Pathol. 63, 559, 1975.

Arch. Surg.

Vol.lO,No.2

HEPARIN AND PLATELET AGGREGATION

2?9

20.

WlJ,K.K. and HOAK, J.C. Spontaneous platelet aggregation in arterial insufficiency; Mechanisms and implications. Proc. Am. Sot. Hematol. t90, 1974 (Abstr).

21.

EIKA, C. Inhibition of thrombin-induced aggregation of human platelets by heparin. Stand. J. Haematol. 8, 216, 1971.

22.

EIKA, c. On the mechanism of platelet aggregation induced by hepnrin, protamine and polybrene. Stand. J. Haematol. 9, 248, 1972.

23.

EIKA, C. The platelet aggregating effect of eight commercial heparins. Stand. J. Haematol. 9, 480, 1972.

24.

HAN, P. and ARDLIE, N.G. Heparin, platelets and blood coagulation., Implications for low-dose heparin prophylactic regimens in venous thrombosis. Br. J. Haematol. 27, 253, 1974.

25.

TEIEN, A.N., ODECARD, O.R. and CHRISTENSEN, T.B. Heparin coupled to albumin, dextran, and ficoll: Influence on blood coagulation and platelets, and in vitro duration. Thromb. Res. 7, 273, 1975.

26.

EIKA, C. Platelet refractor sLate induced by I~cparin. Scond. J. Haematol. 9, 665, 1972.

27.

THOMSON, C., FORBES, C.D. and PRENTICE, C.R.M. The potentiation of platelet aggregation and adhesion by heparin in vitro and in vivo. Clin. Sci. Med. 45, 485, 1973.

28.

ZUCKER, M.B. Platelet release caused by centrifugation, ADP, and epinephrine. Proc. 2nd Congress Int. Sot. Thromb. Haemostat. pp. 276, 1971 (Abstr).

29.

YAMAZAKI, H. TAKAHASHI, T. and SINO, T. Hyperaggregability of platelets in thromboembolic disorders. Thromb. Diath. Haemorrh. 34, 94, 1975.