SummaryCollagen cofactor (CCo), an activity of von Willebrand factor (vWF) which increases the rate of adhesion of human fixed washed platelets (FWP) to collagen, was measured in plasma from normal individuals and individuals with von Willebrand’s disease (vWD). CCo in vWD plasma was compared to vWF antigen (vWF:Ag), ristocetin cofactor (RCo), factor VIII (VIII) coagulant activity (VIII:C) and the quantitative bleeding time. There was close correlation between CCo and VIII:C (r = 0.909), vWF:Ag (r = 0.975), and RCo (r = 0.936). However, there was no correlation between CCo and the quantitative bleeding time. Plasma CCo in type IIA vWD was markedly lower than vWF: Ag and the ratio of CCo/vWF: Ag was 0.08, which was less than a mean value of 0.92 in type I vWD. CCo activity in normal plasma was completely inhibited by monoclonal antibody CLB-RAg 201, an antibody that inhibits the binding of vWF to collagen, suggesting that the binding of vWF to collagen is required for the expression of CCo. Furthermore, the partial inhibition of CCo by monoclonal antibody CLB-RAg 35 that inhibits the binding of vWF to platelet in the presence of ristocetin, suggests that CCo is partly mediated through platelet membrane glycoprotein Ib. Large multimers of vWF:Ag in normal plasma were preferentially absorbed by collagen. These studies demonstrate that CCo is another functional activity of vWF and the measurement of CCo may be useful for the detection of new variant forms of vWD.
To determine a role of platelet membrane components on the interaction of platelet-collagen-von Willebrand factor(vWF), several experimental approaches were used. The adhesion of human fixed washed platelets(FWP) to collagen was decreased after the treatment with Serratia marcescens protease(100 ug/ml), but the collagen cofactor activity(COo) of vWF that enhances the adhesion of FWP to collagen was still present after the digestion. Although the platelet adhesion in the absence of normal plasma was not changed by the addition of monoclonal antibody(M-ab) against platelet membrane glycoprotein(GP) IIb/lIIa(1 0E5, BS Coller), the adhesion was decreased by 30-50% after the treatment of the platelets with 10-100 ug/ml anti-GPIb(6D1, BS Coller). The adhesion of FWP to collagen was inhibited by lectins;the adhesion was 58-75% in the presence of 100-400 ug/ml L. culinaris lectin or weat germ agglutinin and the adhesion was nil in the presence of 100 ug/ml Ricinus communis agglutinin I or 200 ug/ml concanavalin A. By the crossed aff ino-immunoelectrophoresis, the binding of GP Ilb/lIIa in Triton-solubilized platelet supernatant to the collagen spacer gel was observed. When CHAPSO solubilized platelet was applied to the collagen column and the fractions containing adhesion inhibitor were eluated by 0.3M NaCl, Mr of 240K, 220K, 21 OK, 116K, 61K, 54K, 50K and 45K proteins were identified besides the proteins which correspond to thrombospondin, GPIb, GP lib or Ilia by SDS-PAGE(7.5% gel, silver stain). GOo in normal plasma was not changed by anti-GPIIb/lIIa but was decreased to 32-38%by anti-GPIb. M-ab against vWF, CLB-RAg 35(van Mourik), that inhibits the binding of vWF to platelet by ristocetin decreased COo in normal plasma by 70% and CLB-RAg 201 (van Mourik) that inhibits the binding of vWF to collagen did completely inhibit the COo in normal plasma. In conclusion, our data suggest that (1) GPIb is partly involved in the platelet adhesion to collagen; (2) the binding of vWF to collagen is required for the expression of CCo; (3) CCo of vWF is partly mediated though GPIb; and (4) several platelet membrane protein(s) besides GPIb or GPIIb/lIIa may be also involved in both the adhesion of platelets to collagen and CCo of vWF.
A. and YOSHIDA, Y. Factor XIII is Not Involved in Human Platelet-Collagen Interaction. Tohoku J. Exp. Med., 1989, 159 (1), [37][38][39][40][41][42][43][44] A role of factor XIII (FXIII) on the interaction of human platelets with collagen was investigated using either formaldehyde fixed-washed platelets (FWP) or nonfixed platelets. The adhesion of FWP to bovine type I collagen was measured by using either an aggregometer or a collagen immobilized glass beads column. The interaction of non-fixed human platelets with collagen was measured with in vitro bleeding time (Thrombostat-4,000), which was performed by passing citrated whole blood through the filter covered with rat type I collagen under the constant shear stress. FWP adhesion to the collagen immobilized column (1,300 p g collagen) was not changed by the addition of commercial FXIII preparation (Fibrogammin) ; the adhesion was 42.7% in the presence of 1% human serum albumin, 42-43% in the presence of 1-2 U/ml of FXIII. The addition of rabbit antibody to FXIII to normal FWP did not change the degree of a dhesion ; 42.3% (1:100 anti-FXIII) and 46.1% (normal rabbit serum). Furthermore, platelets from the patient with congenital FXIII deficiency normally aggregated by bovine collagen and the adhesion of the patient FWP to the collagen was similar to that of normal FWP. Prolongation of partial thromboplastin time and the changes of thromboelastograph of normal plasma were observed after mixing with the collagen, and factor VIII, FXIII and von Willebrand factor were adsorbed by the collagen. The amount of FXIII in normal human plasma bound to collagen was 17, 23 and 54% at the concentration of the collagen 250, 500 and 1,000 p g/ml, respectively. The binding of plasma ristocetin cofactor was not different between normal control and the patient with FXIII deficiency. These data suggest that FXIII is not involved in human platelet interaction with the type I collagen, while FXIII in normal human plasma binds to the collagen. platelet ; collagen ; factor XIII and factor XIII deficiency
-136-To study the effectiveness of serum transfusion in hemophiliacs, in vitro experiments and in vivo trials were carried out. In in vitro experiments, addition of one volume of serum shortened consisently the kaolin added partial thromboplastin time (kaolin PTT) of both hemophilia A and B plasmas. In the former, the shortening was the same as in the control plasma , but the elevation of AHG level was not seen in the mixture, while in the latter the shortening was more marked than in the control plasma and the PTC level was elevated. In in vivo studies, 80 ml of serum was transfused in each of the cases of hemophilia A and B. In the former, kaolin PTT was shortened from 119.0 to 92.0 sec., whereas the level of AHG was not changed. In the latter, kaolin PTT was shortened from 262.0 to 151.0 sec, and the PTC level was elevated from less than 1% normal to 2.8% normal. Serum transfusion is thought to be an effective hemostatic procedure in hemophilia A and especially in hemophilia B.hemophilia A and B.
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