Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C)

Abstract

Activation of platelets and coagulation in vivo was studied in nine patients with hemophilia A and inhibitors to human Factor VIII, prior to and following treatment with porcine Factor VIII (PFVIII; HYATE:C). In addition, six hemophiliac patients were similarly studied after treatment with recombinant Factor VIII (rFVIII). Platelet activation was also examined in vitro using porcine von Willebrand factor (PvWF)-enriched and PvWF-depleted fractions obtained by fractionation of PFVIII. Coagulation was assessed by measuring the concentrations of plasma prothrombin fragment 1+2 concentrations (prothrombinase generation) and Factor Xa-ATIII. Patients treated with PFVIII had signi®cantly increased numbers of circulating platelets expressing CD62 and CD63 (markers of platelet activation) and annexin V (marker of platelet procoagulant activity) compared to patients treated with rFVIII; the former patients also demonstrated an increase in plasma coagulability after therapy. In in vitro experiments it was observed that the platelet-activating and procoagulant capacity of PFVIII resided in the PvWF-enriched fraction, and the same was true for the plasma hypercoagulability following exposure of platelets to PFVIII. These results support the hypothesis that PFVIII-induced platelet activation provides a mechanism for enhancing hemostasis, separate from, and additional to, that due to increased circulating Factor VIII, and it is due to residual PvWF in the PFVIII preparation. Am.

Porcine factor VIII

“…Porcine VWF binds to the glycoprotein Ib receptor on platelets, leading to activation of the glycoprotein IIb/IIIa receptor, which in turn causes to platelet aggregation associated with binding of fibrinogen [18]. Flow cytometry also demonstrated activation of platelets following treatment with Hyate:C, reflected by an increase in the number of circulating platelets expressing CD62 and CD63 and annexin V [19]. The authors of this study speculated that the platelet activation caused by infusion of porcine factor VIII enhanced haemostasis through a quite separate, but complementary pathway to that simply because of increased circulating factor VIII.…”

The Relationship of von Willebrand Factor Binding to Activated Platelets from Healthy Neonates and Adults

“…Flow cytometric analysis was performed as described elsewhere (30,33). In brief, 5-L aliquots of unfixed or 10 L of fixed blood taken into CTAD were incubated with 5 L of MAb and 50 L of buffer [145 mM NaCl, 5 mM KCl, 1 mM MgCl, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), containing 2.0 mM Mg ϩϩ ].…”

“…To evaluate the ability of activated platelets from adults to bind VWF, citrated-475 VWF BINDING TO PLATELETS FROM NEONATES whole blood was exposed to thrombin (0.1 or 1 U/mL) or ADP (5 or 10 M) before fixation and staining, as described elsewhere (30,33). In brief, 45 L of citrated blood was diluted 1:1 with buffer, incubated with 2.5 mM GPRP and thrombin or ADP for 10 min at 37°C, and then fixed with 1% PF for 10 min.…”

“…In neonates 2-3d and adults, platelet-bound VWF correlated with activation of platelets as measured by CD62P expression. Because different mechanisms of platelet activation may be reflected by different markers of platelet activation (33,38,39), we also tested other markers of platelet activation in the four groups of subjects examined. As shown in Table 3, there was no significant difference in activation status of unstimulated platelets from the control groups of children and adults.…”

von Willebrand factor (VWF)-dependent human platelet activation: porcine VWF utilizes different transmembrane signaling pathways than does thrombin to activate platelets, but both require protein phosphatase function

“…As a model of VWF‐dependent platelet activation, we have employed porcine VWF (pVWF), which is able to functionally bind to human platelets via a molecular domain closely related with that of human VWF [18]. Porcine VWF (as is true for cell‐associated human VWF or human VWF + ristocetin) induces platelet agglutination [16,17,19] and initiates platelet transmembrane signaling [16,17,20]. Our results indicate that pVWF‐induced platelet activation, including platelet agglutination and microparticle formation, required the activity of protein tyrosine and serine/threonine phosphatases.…”