Thrombin-mediated Feedback Activation of Factor XI on the Activated Platelet Surface Is Preferred over Contact Activation by Factor XIIa or Factor XIa

Frank A Baglia,Peter N Walsh

doi:10.1074/jbc.m000464200

Abstract

To study the pathways for initiation of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as surfaces for factor XI activation by factor XIIa, factor XIa, or thrombin. Activated gel-filtered platelets promoted the activation of factor XI (60 nm) by thrombin (0.02-10 nm, EC(50) approximately 100 pm, threshold concentration approximately 10 pm) at initial rates 2- to 3-fold greater than those obtained with dextran sulfate in the presence of either high molecular weight kininogen (45 nm) and ZnCl(2) (25 micrometer) or prothrombin (1.2 micrometer) and CaCl(2) (2 mm). The maximum rates of factor XI activation achieved in the presence of activated gel-filtered platelets were 30 nm.min(-1) with thrombin, 6 nm.min(-1) with factor XIIa and 2 nm.min(-1) with factor XIa. Values of turnover number calculated at various enzyme concentrations (0.05-1 nm) were 24-167 (mean = 86) min(-1) for thrombin, 4.6-50 (mean = 21) min(-1) for factor XIIa, and 1.3-14 (mean = 8) min(-1) for factor XIa. A physiological concentration of fibrinogen (9.0 micrometer) inhibited factor XI activation by thrombin (but not by factor XIIa) in the presence of dextran sulfate but not in the presence of gel-filtered platelets. Compared with factors XIIa and XIa, thrombin is the preferred factor XI activator, and activated platelets are a relevant physiological surface for thrombin-mediated initiation of intrinsic coagulation in vivo.

Highlights

Human coagulation factor XI is a disulfide-linked homodimer consisting of two identical polypeptide chains each containing 607 amino acids
The Activation of Factor XI by Thrombin in the Presence of Activated Platelets or Dextran Sulfate—We previously determined that activated platelets in the presence of added high molecular weight kininogen (HK) (45 nM), ZnCl2 (25 ␮M), and CaCl2 (2 mM) could promote the activation of factor XI by thrombin at initial rates 2- to 5-fold greater than those obtained in the presence of dextran sulfate [15]
Effects of Fibrinogen on Factor XI Activation—We have demonstrated that HK (45 nM) is a cofactor that facilitates factor XI binding to platelets and optimal rates of factor XIa formation with platelets [15]

Summary

EXPERIMENTAL PROCEDURES

Materials—Human prothrombin, human factor XIIa, and human factor XIa were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Incubations were carried out at 37 °C in 200 ␮l of Tris (50 mM), NaCl (150 mM), pH 7.3, with 1% bovine serum albumin and dextran sulfate (1 ␮g/ml, average Mr ϭ 500,000; Sigma) or gel-filtered platelets (activated by incubation at 37 °C for 1 min with the thrombin receptor activation peptide (SFLLRN-amide, 25 ␮M), or 2 ␮M phospholipid vesicles (consisting of phosphatidylserine and L-␣-dioleoyl-phosphatidylchlorine, 1:3 ratio), prepared as described previously [32]. The amount of factor XIa generated was assayed by reference to a standard curve constructed using purified factor XIa. Initial rates were obtained by visual estimation during the linear portion of the progress curves by dividing factor XIa formed (nanomolar) by the time (minutes): 2 min for the enzymes thrombin and factor XIIa and 15–20 min for factor XIa at various concentrations.

RESULTS

No surface

DISCUSSION

ADDITIONS AND CORRECTIONS