Low Molecular Weight Heparin-Catalyzed Inactivation of Factor Xa and Thrombin by Antithrombin III – Effect of Platelet Factor 4

Abstract

SummaryLow molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding material, so mean molecular weights as such might bear little information on their anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities by platelet factor 4 (PF4). These properties were investigated in pure systems with proteins of human origin. Pseudo-first order rate constants of inactivation of factor Xa and thrombin by antithrombin III were determined as function of heparin concentration, in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor Xa over the anti-thrombin activities were essentially the same for the 4th International Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32), CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed that the heparin molecules of the upper region of the molecular weight distribution are responsible for the anti-thrombin, but also to a large extent for the anti-factor Xa activities. The results indicate that depolymerization of unfractionated heparin does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the presence of Ca2+-ions the rate constants of inactivation of factor Xa are lowered as compared to those of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin activities of fixed concentrations of the LMW heparins was studied by measuring rate constants as function of PF4 concentration. All anti-thrombin and 50% of the anti-factor Xa activities were readily neutralized. Excess PF4 was required to neutralize another 35-50% of the anti-factor Xa activities. At PF4 levels obtained at maximal release of the content of platelet α-granules, all anti-thrombin and most (≥85%) of the anti-factor Xa activities can be neutralized.

Effect of the anti‐factor Xa and anti‐factor IIa activities of low‐molecular‐weight heparins upon the phases of thrombin generation

“…This may be because the laboratory test used for the measurement of anti-FXa activity is performed in platelet-poor plasma (PPP). Platelet factor 4 (PF4) released from activated platelets partially neutralizes some of the anticoagulant activity of LMWHs, albeit to a lesser extent than UFH [18][19][20][21]. Thus, the measurement of anti-FXa activity in plasma does not necessarily reflect the interaction of LMWHs with plasma and cellular components of blood coagulation.…”

Von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate

“…Human ␣-thrombin was purified as described. 19 D-Phe-ProArg chloromethylketone (PPACK) came from Calbiochem (San Diego,…”

“…Human ␣-thrombin was purified as described. 19 For personal use only. on May 11, 2018. by guest www.bloodjournal.org From CA).…”

Regulation of Platelet Factor Va-dependent Thrombin Generation by Activated Protein C at the Surface of Collagen-adherent Platelets

“…Native type I collagen fibrils were extracted from bovine Achilles tendon in the absence of proteases using 0.5 M acetic acid and precipitated with 1.7 M NaCl as described (12). Human ␣-thrombin was prepared as described previously (13). Human activated protein C (APC) was purchased from Kordia (Leiden, The Netherlands).…”