Analytical and Clinical Validation of a Non-Ristocetin Based VWF Assay on 2 Automated Analyzers in a Large Reference Laboratory.

Background: Historically, von Willebrand factor (VWF) activity assays utilized ristocetin despite limitations including poor limits of detection and high imprecision. Newer VWF activity assays such as the INNOVANCE® VWF Ac assay, however, do not rely on ristocetin to measure platelet-dependent VWF function. The purpose of this study was to evaluate the analytical and clinical performance of the Siemens Healthineers INNOVANCE VWF Ac Assay on the Siemens BCS® XP and the Sysmex® CS-2500 systems in a large reference laboratory setting.

Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.

Thrombin-activated factor Va exists as two isoforms, factor Va(1) and factor Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells.

Monocyte/macrophage regulation of coagulant events.

Monocytes/macrophages actively regulate both the assembly and function, as well as the substrate specificity, of various coagulation enzymes at their membrane surface. Regulation is effected through a variety of mechanisms, not limited to, but including the expression of receptors (or 'binding sites') for the various protein constituents of the complexes, the expression of different receptors which may alter the function of the protease, and the expression of membrane proteases which may affect protein cofactor function. Monocyte stimulation with various agonists modulates many of these responses as does their adherence to and differentiation on various substrates.

Procoagulant activities expressed by peripheral blood mononuclear cells.

These combined data support the concept that the procoagulant response elicited by mononuclear cells, particularly monocytes, is accomplished through regulated binding site-mediated (or perhaps "receptor"-mediated) assembly of proteolytic activities at their membrane surface. Because the work of several laboratories indicate that the monocytes provide the appropriate membrane surface for the assembly and function of all the coagulation complexes required for thrombin production in vivo, monocytes may provide a unique opportunity to investigate how coagulant reactions are regulated on cell surfaces through both receptor-mediated events as well as by channeling a product of one reaction to serve as a mediator of a second reaction.

Factor Xa interacts with two sites on monocytes with different functional activities.

Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration.

Endotoxin enhances the expression of monocyte prothrombinase activity.

Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents. Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.

The detection of platelet alloantibodies by flow cytometry.

A flow cytometric procedure was investigated for its ability to detect antibodies directed against blood group A, HLA, and PlA1 (HPA-1a) antigens. When type O sera were tested against platelets from blood group A donors, only 9 of 14 positive reactions were observed. Furthermore, the expression of blood group A varied more than 100-fold on platelets derived from individual donors.