Summaryvon Willebrand factor (vWF) mediates platelet adhesion at sites of vascular damage. It acts as a bridge between receptors on platelets and collagens present in the connective tissue. Two collagen binding sites have been identified on the A1 and A3 domain of the vWF subunit. To study the functional importance of these binding sites, we have made two deletion mutants that lack the A1 domain (residues 478-716; ΔA1-vWF; Sixma et al. Eur. J. Biochem. 196,369,1991 [1]) or the A3 domain (residues 910-1113; ΔA3-vWF). After transfection in baby hamster kidney cells overexpressing furin, the mutants were processed and secreted efficiently. Ristocetin or botrocetin induced platelet binding was normal for ΔA3-vWF as was binding to heparin and factor VIII. As reported by Sixma et al. (1) ΔAl-vWF still binds to collagen type III, indicating that the A3 domain is sufficient for the interaction. In the current study, we investigated the binding of ΔA3-vWF to collagen type III. When preincubated on collagen type III it did not support platelet adhesion under flow conditions, whereas it was able to support platelet adhesion when coated directly to a glass surface. The binding of 125I-ΔA3-vWF to collagen was specific but maximal binding was about 40 times less compared to 125I-vWF. When added at 25 times excess, ΔA3-vWF did not compete with 125I-vWF for binding to collagen type III, whereas ΔAl-vWF did. The binding of 125I-ΔA3-vWF could be blocked by excess unlabeled vWF but not by ΔA1-vWF. In conclusion, we demonstrate that the A3 domain in vWF contains the major collagen binding site. The major binding site present on the A3 domain and the minor site present on A1 bind to different sites on collagen.

lntroduction a more gradual drop at higher shear rates above 2000/sec. Aggregates were larger in the single pass system. Measurements of B-thromboglobulin in the outflow of the chamber showed a slight but significant increase in the recirculating system indicating that more platelet activation occurred in this system. The difference between the single pass and the reperfusion system was not observed for collagen type I and III. The role of divalent cations in adhesion to collagen type IV was compared to that of adhesion to collagens type I and III and to endothelial cell marix. Adhesion at 1600/sec was very sensitive to the Mg'* concentration for collagen type IV whereas little effect was seen at 300/sec. Also platelet adhesion to endothelial cell matrix and to collagen type III were sensitive to the Mg'* concentration at 1600/sec but the effect was less than for collagen type IV. Adhesion to collagen type I was not very sensitive to Mg2*. Aggregate formation on collagen type IV and I was strongly impaired at low Mg'*, but aggregates on collagen type III were almost not affected. These results indicate that variations in the physiological range of Mg2* may have an effect on platelet adhesion in vivo. The Mg'* concentration may have an effect on bleeding from wounds. Platelet adhesion to collagen type VI was much stronger at 100/sec (60 Vo coverage) than at 1000/sec (6Vo) (6). In contrast to the study by Saelman, however, aggregation at 100/sec was stronger for collagen type VI than for collagen type I (4).This may be due to differences in the collagen type VI, that was used. Ross et al used collagen type VI purified from umbilical cords in which the globular domains were intact. Another difference was the use of a single pass system instead of the recirculating system of Baumgartner e[ al. that was used in the earlier study. Adhesion to collagen type VI required von Willebrand Factor (vWF) and was inhibited by antibodies to GPIb and by aurin tricarboxylic acid. More unexpected was the observation that an antibody to GPIIb-IIIa was inhibitory not only for platelet aggregation but also almost completely abolished adhesion to collagen type VI whereas less effect was seen on adhesion to collagen type I. It is important to note that these studies were performed in citrated blood. The effect of GPIIb-IIIa inhibition on adhesion to collagen type I is strong in citrated blood and almost absent in heparinized blood. Collagen types Platelet adhesion to collagen is an essential first step in haemostasis and thrombosis. After platelets have adhered they become activated, other platelets stick to the adhering platelets and a haemostatic plug or platelet thrombus is formed.

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Blood coagulation functions as part of the innate immune system by preventing bacterial invasion and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca 2+ -and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) † Corresponding author. o-donnellvb@cardiff.ac.uk (V.B.O'D.); collinspw@cardiff.ac.uk (P.W.C.). * These authors contributed jointly to the work. Author contributions:Experiments were conducted by SNL, DAS, GM, RU, AOC, DF, JM, SR, VJT, AB, SF, MA, MH, KAR, CPT, JA and GK, and designed by SNL, DAS, PDG, SH, VBO, SAJ, PRT, PWC, PVJ. CLP and SO provided clinical samples. AP provided supervision and training. SNL, DAS, VBO and PWC wrote the paper. All authors edited the manuscript. Competing interests:The authors have declared that they have no competing interests. Europe PMC Funders GroupAuthor Manuscript Sci Signal. Author manuscript; available in PMC 2017 December 07. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein β2GPI (β2-glycoprotein I), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.