Von willebrand factor, polycations, and platelet agglutination

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VON WILLEBRAND FACTOR, POLYCATIONS, AND PLATELET AGGLUTINATION

Terry K. Rosborough Section of Hematology,Departmentof Medicine MinneapolisVeterans AdministrationMedical Center and Universityof Minnesota (Received

ABSTRACT

20.8.1.979; in revised Accepted by Editor M.A.

form 5.11.1979 Packham)

In the presence of ristocetin,factor VIII-relatedvon Willebrand factor (VIIIR:WF)causes platelet agglutination. This report shows that several synthetic polycationsalso participatewith VIIIR:WF to cause platelet agglutination;one species of polyornithine,two species of Polybrene, and four species of polylysinecause macroscopic platelet agglutinationthat is mwch stronger in the presence of normal human plasma than it is in the presence of von Willebrand four polycatfonichistones and four disease plasma. In contrast, lectins induce identical platelet agglutinationreactions in the presence of both normal and von Willebrand disease plasma. Conunercial factor VIII concentratecontains both ristocetincofactor and the cofactor for the synthetic polycations. The sucrose density gradient sedimentationpatterns of these two cofactors are indistinguishable. These results show that a variety of agents can detect a VIIIR:WF deficiency in human plasma. These studies do not indicate whether the ristocetin cofactor and the polycation cofactor are identicalor only closely related activities.

INTRODUCTION During many platelet reactions, platelet adhesion to a surface is an important early event (12). The extent of platelet adhesion to a surface is controlled by many complex factors, including the concentrationof some noncellular elements in the blood (2,3). Factor VIII-relatedvon Willcbrand factor (VIIiR:WF)is an important non-cellularblood element that augments platelet adhesion to surfaces (3-6). In addition to its vital role in physiOlOgiChemostasis,VIIIR:WF may also vessel walls (7,8).

participate

in processes that damage

Key Words: Von Willebrand disease, factor VIII, polycations,ristocetin, platelet agglutination 481

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The study of the platelet-VIIIR:WF interaction was advanced by the discovery that, in the presence of VIIIR:WF, the cationic antibiotic ristocetin caused platelet agglutination (9,lO). Despite much investigation, however, the mechanism of the ristocetin-platelet-VIIIR:WF interaction is incompletely understood. Lalezari and Spaet (11) found that Polybrene (hexadimethrine bromide), a well characterized synthetic polycation, would directly agglutinate platelets. I recently found that Polybrene-induced platelet agglutination is augmented by a factor that is deficient in VWD plasma (12) and this study shows that many, but not all, platelet-agglutinating polycations will detect a very similar deficiency in VWD plasma. MATERIALS.ANO METHODS Plasmas: Platelet-poor plasma was prepared by 50G3 g, 15 min, centrifugation of blood anticoagulated with 0.011 M sodium citrate. A reference normal pool was made from plasma of eight healthy male and female adults. Plasma from three patients (not recently transfused) with severe VWD was used in these studies. All three plasmas contained no detectable ristocetin cofactor or electroinnnunoassay-determinedfactor VIII-related antigen (13). Plasma TD, from a young adult male with congenital VWD, was kindly supplied by Dr. Roger Edson, University of Minnesota, Department of Laboratory Medicine. Plasma GK, from a young adult female with congenital VWO, was purchased from George King Biomedical, Inc., Salem, NH. Plasma VC was donated by an elderly male patient at the Minneapolis V.A. Medical Center who was recently described because of the probable acquired nature of his disease, even though no inhibitor of ristocetin cofactor was found in his blood (14). Fixed, washed platelets (FWP): FWP were prepared by fixing 24 hour-old blood bank platelet concenhrates in an equal volume of 2% paraformaldehyde solution for 18 hours at 4 C (12,15). After two washings, the FWP were suspended in 0.03 M tris(hydroxymethyl)aminomethane-buffered.0.14 M sodium chloride, pH 7.4 (tris-saline) to a concentration of 600,000 per microliter. Each preparation of FWP was preserved with 0.01% sodium aride and was discarded when two weeks old. Platelet agqlutinating aqents: Ristocetin was purchased from Bio-Data Corporation, Willow Grove, PA Polybrene (PB) lots 050127 and 011777 was purchased from Aldrich Chemical Company, Milwaukee, WI. The osmolality of 50 mg/ ml solutions (in demineralized water) of each PB lot was measured by freezing point depression using a 4DII Osmometer (Advanced Instruments, Inc.). The lot 050127 solution contained 115 milliosmoles/Kg, while the lot 011777 solution contained 133 milliosmoles/Kg. Poly-L-lysfne (PL) types II (mol. wt. 3000), V (mol. wt. 15,000), VA (mol. wt. 30,000), VII B (mol. wt. 60,000), and 16 (mol. wt. 75,000) were all purchased from Sigma Chemical Company, St. Louis, MO. Poly-L-alpha ornithine (PO) type IC (mol. wt. 270,000) and histones types II, IIIS, V, and VIIIS were also purchased from Sigma Chemical Co. The lectins Ricinis cotnnunis120, phytohemagglutinin, and wheat germ agglutinin were all purchased from Miles Laboratories, Elkhart, IN. All platelet agglutinating agents were dissolved in 0.03 M tris-saline and adjusted to pH 7.4. Ristocetin cofactor (RCF) assay_: RCF was measured using a macroscopic tilt-tube method (12,15) that was modified so that all test samples contained bovine albumin (Sigma Chemical Co.) in a final concentration of approximately 5 mg/ml, as suggested by Stibbe and Kirby (16). in duplicate at several sample dilutions.

All test samples were assayed

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lleasurementof FWP agglutination induced by poiycations and iectins: Agglutination of FWP by PL, PO, PB, histones and lectins was measured with a macroscopic tiit-tube method similar to that used to measure RCF. An agglutination endpoint was called only when a definite, readily visible agglutination reaction had occurred. In sore test reactions, a very small amount of FWP agglutination was seen with close inspection, but the response never proceeded to a complete endpoint. For such reactions, the time of the incompiete agglutination response was recorded, but was qualified with a + symbol. These t reactions, when examined by phase microscopy, contained smaller FWP agglutinates than definitely positive endpoint reactions. Each FWP agglutination reaction, that was performed in the presence of plasma, was accompanied by an appropriate control reaction to ensure that plasma precipitation was not occurring. All the reported FWP agglutination times are mean values of at least four determinations. The precision of this macroscopic method for detecting FWP agglutination was studied by performing multiple replicates with several different polycations in the presence and absence of plasma; in all these studies, the coefficients of variation (SD/mean x 100) were less than IO%. To achieve this precision, experience and careful technique were necessary. Polybrene cofactor (PBCF) assay: PBCF was measured with the macroscopic tilt-tube assay: 0.2 ml FWP, 0.1 ml of tris-saline, 0.05 ml of severe ViiD plasma, 0.05 ml of test plasma appropriately diluted in VWD plasma, and 0.3 ml of PB (lot 050127, 0.1 mg/ml final concentration) were mixed and timed for agglutination. The standard curve for this assay was generated using dilutions of the normal human plasma pool and was plotted linearly on log-log graph paper. The pooled normal plasma was considered to contain 100 units(U)/dl of PBCF. The lower limit of sensitivity of this assay was approximately 5 U/dl. The coefficients of variation for multiple replicates of the standard curve were less than 10% for each plasma dilution from 100% to 5%. Density gradient ultracentrifugation of factor VIII concentrate: Fivetenths of a ml of reconstituted Hemophil (Hyland Laboratories, Costa Mesa, CA) were layered over 12.0 ml linear 5-40% sucrose gradients. The gradients were centrifuged at 160,000 x g-tr,ax for 16 hours at 15 C using a SW-41 rotor in a Sorvall OTD-2 ultracentrifuge. 1.25 ml gradient fractions were collected using upward flow elution (10 fractions per gradient). The protein concentration in each gradient fraction was measured with the Lowry technique using human albumin as a standard (17). The RCF and PBCF concentrations in each gradient fraction were measured-by testing the ability of 0.1 ml of each fraction to correct the RCF and PBCF deficiency in 0.9 ml of severe human VWD plasma. RESULTS In the absence of plasma, ail of the synthetic polycations aggiutinated FWP with a potency that varied directiy with their molecular weights (Table 1) The three largest and most potent polylysines produced weaker FWP agglutination at higher concentrations than they did at lower concentrations. The molecuiar weights of the two Poiybrene lots are unknown, but the platelet agglutinating activity of the two lots clearly differed. According to Aldrich Chemical Co., the two lots were produced by the same method, but lot 050127 is several years older than lot 011777. The lower osmolality of the of PB lot 05Oi27 compared to the solution of lot Oil777 (see platerials and Nethods) suggests that lot 950127 is a larger polymer than lot

Solution

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011777, consistent with the relative strengths of the two PB lots as FWP agglutinating agents.

TABLE 1 Agglutination of FWP by PB, PL, and PO in the Absence of Plasma Polycation Final Concentration .(pg/ml)

2000 Polycations

Molecular Weight

1000

100

5

2.5

1.25

Agglutination Times (Set)

Pa-777

*

10

10

17&

>60

~60

>60

Pa-127

*

10

7

7

>60

>60

>60

3 x lo3

10

182

>60

>60

>60

~60

PL-v

15 x lo3

7

6

6

~60

>60

>60

PL-VA

30 x lo3

6

6

6

>60

>60

~60

PL-VIIB

60 x lo3

>60

6

6

9

>60

~60

PL-IB

75 x lo3

~60

6

6

8

>60

>60

PL-IC

270 x lo3

>60

6

6

7

12

>60

PL-II

* = unknown + = incomplete reaction

Each of the synthetic polycations produced weaker FWP agglutination in the presence of normal titrated plasma than in the presence of tris-saline (Table 2). The normal plasma weakened the polycation agglutinating activity in part due its albumin content, since purified human albumin (Sigma Chemical Co.) alone inhibited polycation induced FWP agglutination. Another plasma inhibitory effect was the citrate anticoagulant, since 0.011 M titrated normal plasma caused weaker polycatfon-induced FWP agglutination than either 0.068 M EDTA or 0.2 unit/ml heparinized plasmas. For each polycation except the smallest (PL-II),a narrow concentration range existed that caused an FWP agglutination reaction that was much stronger in the presence of normal plasma than in the presence of any of the three VWD plasmas (Table 2). If too little or too much of each polycation was added, the normal and the VWD plasmas could not be distinguished,

Al-

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thougq all three VWD plasmas contained no detectable RCF activity, they demonstrated slightly different responses with the different polycations. The four polycationic histones and the four lectins also strongly agglutinate FWP in the absence of plasma (not shown). In contrast to PB, PL, and PO, however, the histones and lectins, over a wide range of concentrations, produced identical platelet a glutination times in the presence of both normal and VWD plasma (not shown7 .

TABLE 2 Agglutination of FWP by PB, PL, and PO in the Presence of 20% Normal and VWD Plasma Plasmas

Normal Polycations

VWD-GK

VWD-VC

Agglutination Times (Set)

PB-777

2000

13

PB-127

100

10

4000

>60

PL-v

400

12

172

16

25+-

PL-VA

400

12

162

16

26k

PL-VIIB

90

11

20+

17*

30t

PL-IB

90

11

19+

15

3Ozk

PO-IC

80

9

16

15

252

PL-II

2

VWD-TD

>60 25+ >60

>60 25+ >60

>60 22+ >60

= incomplete reaction

Hemophil, which contained both RCF and PBCF in approximately equal concentrations, was ultracentrifuged through a linear sucrose density gradient and the protein, RCF, and PBCF concentrations in each gradient fraction were measured. The RCF and PBCF assay standard curves for these studies were generated in the presence of sucrose, since sucrose modestly inhibited ristocetin-induced FWP agglutination. Figure 1 shows that RCF and PBCF sedimented indistinguishably as species with high sedimentation coefficients. The gradient fractions containing the peak RCF and PBCF activity also corrected the PL and PO cofactor deficiencies in the three VWD plasmas.

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FIGURE 1 Sucrose Density Gradient Sedimentation of Hemophil

o---dProtein ConcentrOtion o----aPBCF Concentration m-=-o

RCF Concentration

-9

-0 -7 -6 -5 -4 -3 -2

I

.’

ti

I

#

2

4

6

8

IO

8.

Fraction Number DISCUSSION The mechanism of the platelet-VIIIR:WF interaction is not understood. Much of our knowledge about this important interaction comes from studies of ristocetin-induced platelet agglutination. Some of these studies have suggested that ristocetin induces the human platelet-human VIIIR:WF interaction primarily by altering the VIIIR:WF, causing it to bind to human platelets just like unaltered bovine factor VIII binds to human platelets (18-21). A second major hypothesis for the ristocetin-VIIIR:WF-platelet interaction is that the positively charged ristocetin molecule binds to the negatively charged platelet surface, resulting in an alteration that permits unaltered human VIIIR:WF to bind to specific sites on the platelet surface (22). I have investigated various cationic substances to see if positively charged agents other than ristocetin can also induce the platelet-VIIIR:WF interaction. consistent with previous reports, I have found that the synthetic polycations Polybrene, polylysine, and polyornithine can all induce direct -piatelet agglutination in the absence of any plasma components (11,23-27). My *finding that the platelet agglutinating potency of these synthetic polycations varies directly with their molecular weight agrees with most, but not all previous reports (23-26). The larger polycations may have greater agglutinating potency either because they bind more effectively to the platelet surface, resuiting in a greater reduction in platelet surface charge, or because they have a greater effective length, resulting in a greater ability to bind

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simultaneously to the surface of two neighboring plateiets. My finding of paradoxically weaker FWP agglutination with the highest concentrations of the three largest polylysines was also reported by Kirby and Mills (25) and may result from the repulsive effect of positive piatelet surface charges that could develop if larger polycations do bind more effectively to the platelet surface. All of the syntnetic poiycations prociuceaweaker piateiet agglutination in the presence of normal titrated plasma than in the presence of buffer. The inhibitory effect of citratea plasma was due in part to aibumin, which may bind the polycations and make them unable to react with the FWP. A similar albumin effect has been described for ristocetin induced FWP agglutination (16) and Guccione et al. reported that iow concentrations of aibumin reduced the aggregation and release reactions of washed, iive platelets in response to polylysine (27). An additional polycation inhibiting substance in plasma is the citrate anticoagulant. Anticoagulant concentrations of EDTA and heparin do not have a similar inhibitory effect. Citrate does not inhibit ristocetininduced platelet agglutination, perhaps because ristocetin is less positively charged than PL, PO, or PB and therefore less able to bind to citrate. Regardless of their strengths as direct platelet aggiutinating agents, each of the synthetic polycations, except for the smallest and weakest (PL II), produced greater platelet agglutination in the presence of normal plasma than it produced in the presence of VWD plasma. To produce this differential response, each polycation had to be added in a critical concentration to the plasma-FWP mixture; too little or too much polycation would not distinguish the norrilal from the VWD plasma. Too much polycation may cause such strong direct platelet agglutination that the effect of any polycation cofactor is overwhelmed, while too little polycation probably cannot activate the polycation cofactor-FWP interaction. Even though the lectins and the polycationic histones in this study also could directly agglutinate platelets, they could not distinguish normal plasma from VWD plasma. Thus, not all polycations or all direct platelet agglutinating agents can react with VIIIR:WF to augment macroscopic FWP agglutination. Both RCF and the polycation factor [measured as PBCF) are present in commercial factor VIII concentrate. During sucrose density gradient ultracentrifugation, both cofactors sediment indistinguishably as high molecular weight substances, as described by others for RCF (28,29). Although fibrinogenfibrin complexes may also sediment as high molecular weight complexes, PBCF cannot be related to these complexes, since I have found that PBCF concentration is the same in titrated plasma and titrated serum. PBCF is also.unlikely to be related to cold insoluble globulin (fibronectin), since unlike PBCF, cold insoluble globulin is normal in VWD plasma (30). Coller recently confirmed that Polybrene induces a platelet-VIIIR:WF interaction similar to that induced by ristocetin (31). My studies suggest that RCF and the polycation cofactor are closely related, though not necessarily identical, activities. Although Coller concluded that his Polybrene studies supported his hypothesis for the induction of the platelet-VIIIR:WF interaction (22), I do not think my studies yet definitely indicate whether polycations induce the VIIIR:WF-plateiet interaction by affecting the platelet surface or by aitering VIIIR:WF. My studies do show that substances that induce this important interaction are more common than has been suspected. Read et al. recently reported that.a group of snake venoms could also interact with VIIIR:WF (32). The relationship of these venoms to polycations or ristocetin is not yet known.

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ACKNOWLEDGEMENTS The technical assistance of Nancy Carsberg and Tracy Husing was much appreciated in the preparation of this paper. This work was supported by a grant from the Veterans Administration.

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