Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) and Cryoprecipitated AHF Contain Von Willebrand Factor with Comparable Binding to Collagen and Support Shear-Induced Platelet Thrombus Formation

Abstract

Traumatic hemorrhage is a leading cause of morbidity and mortality and is a potentially preventable cause of death. Trauma-induced coagulopathy (TIC) exhibits complex pathophysiology. Fibrinogen (FIB) deficiency, a major mediator of TIC, is an independent predictor of mortality; and one of the first factors to reach critically low levels during hemorrhage. Early supplementation of FIB using cryoprecipitated AHF (Cryo) administration in resuscitation has been shown to improve outcomes in the treatment of TIC. Unlike FIB concentrates, Cryo prepared from conventional plasma is enriched in von Willebrand factor (VWF), factor VIII, factor XIII, and fibronectin that are impacted by TIC in addition to FIB. However, Cryo has a limited post thaw shelf-life of 4-6 hours at room temperature. This causes logistical impediments for early clinical application in trauma often resulting in use late in resuscitation with significant wastage. Pooling of individual Cryo units to achieve therapeutic doses increases donor exposure with risk of bacterial and viral transfusion transmitted infections (TTI). FDA approved Pathogen Reduced Cryoprecipitated Fibrinogen Complex (IFC) manufactured from Amotosalen/UVA treated plasma (PR plasma) is an alternative FIB source that has a 5-day post-thaw shelf-life at RT with reduced TTI risk. Due to its longer post-thaw shelf-life, it can be thawed in advance so that it is readily available and can be administered early in massive transfusion protocols with significantly lower wastage. VWF is a key component of Cryo and plays a major role in hemostasis during resuscitation with TIC. Here we have compared VWF contained in Cryo and IFC in various functional assays. At equimolar concentrations as assessed by measured VWF antigen, IFC and Cryo supported similar ristocetin-induced platelet aggregation. Accordingly, VWF multimeric composition, particularly presence of the largest molecular species, was similar in IFC and Cryo. We then evaluated the ability of VWF in IFC and Cryo compared to native blood to support platelet adhesion and platelet-rich thrombus formation onto a fibrillar type I collagen surface exposed to a linearly increasing shear rate gradient (0 t0 20,000 s -1) in the direction of flow from inlet to outlet of a Hele-Shaw flow chamber. Briefly, human blood, collected in EDTA, Lepirudin and supplemented with PGE 1 was centrifuged to separate blood cells from plasma. Red cells and platelets were washed to remove all plasma VWF and reconstituted to original hematocrit with Tyrode buffer at pH 7.4 spiked with either Cryo or IFC up to 1 U/ml of VWF. Platelets in native blood and in the different mixtures were fluorescently labeled with 3,3'-dihexyloxacarbocyanine iodide (DiOC 6) for visualization, and image sequences were acquired at different shear rates along the gradient. Images were analyzed to quantify platelet adhesion (the proportion of the field of view covered by platelets) and aggregation (i.e., the number and area of aggregates) at any given shear rate. As a negative control, blood reconstituted with buffer instead of a VWF source was used to ascertain that plasma VWF was effectively removed. As positive control, normal donor blood was used without washing out VWF (native blood). At all tested shear rates, the total surface coverage (1 pixel = 0.099 µm 2) by platelets was comparable with VWF in AHF or IFC added to washed blood and native blood (Figure). Of note, the individual size of platelet aggregates tended to increase with increasing shear rates and the number of individual aggregates tended to decrease. Specific monoclonal antibodies against VWF A1 and A3 domains - blocking interaction with GPIbα and collagen, respectively - markedly reduced the number and size of platelet aggregates over the surface. These results indicate that VWF derived from IFC, comparable to Cryo and native blood, retains functions strictly required to support platelet adhesion and aggregation at extremely elevated levels of shear rate/stress. The figure shows the size where 1 pixel = 0.099 µm 2 of each individual platelet thrombus on the surface.