Tze-Chein Wun

The Interaction Between LACI and Heparin

Lipoprotein-associated coagulation inhibitor (LACI) is a multivalent protease inhibitor that directly inhibits factor Xa and, in a factor Xa dependent fashion, inhibits the factor VIIa/Tissue Factor (TF) catalytic complex (1). Its structure is unique: An acidic amino-terminal domain is followed by three tandem Kunitz-type protease inhibitor domains and a basic carboxyl-terminal region (Fig. 1) (2). The Kunitz-2 domain in LACI is needed for inhibition of factor Xa, while the Kunitz-1 domain is required for the inhibition of the factor VIIa in a factor Xa-LACI-factor VIIa/TF quaternary inhibitory complex (3). The function of the Kunitz-3 domain in LACI is not known. In human blood, LACI circulates predominantly in association with the plasma lipoproteins but it is also carried in platelets which release LACI following stimulation by thrombin (4, 5). A third pool of LACI is thought to be bound to glucosaminoglycans at the surface of the endothelium and is released into plasma following the infusion of heparin in vivo (6, 7).

Crystallization of a chymotrypsin inhibitor from Erythrina caffra seeds

Crystals of a chymotrypsin inhibitor from Erythrina caffra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 67.2 A and alpha = 99.4 degrees, and diffract to 3 A resolution.

Heparin-tissue Factor Pathway Inhibitor Complexes: Anticoagulant and Pharmacokinetic Properties

Exogenous addition of full-length tissue factor pathway inhibitor (FL-TFPI) to plasma caused a greater pro longation of prothrombin time (PT) than activated partial thromboplastin time (APTT). In contrast, heparin elicited a greater prolongation of APTT than PT. These results suggest that FL-TFPI and heparin exert their anticoagulant activity pri marily through inhibition of the extrinsic and intrinsic path ways, respectively. Using a dilute thromboplastin-induced clot ting assay, it was found that FL-TFPI was ~37-fold more potent than the carboxyl terminus truncated form (CT-TFPI) in pro longing the clotting time, which indicated that the positively charged carboxyl terminus of FL-TFPI was crucial for its an ticoagulant activity. Both FL-TFPI and CT-TFPI could exert synergistic anticoagulant action with heparin when TFPIs and heparin were added sequentially to plasma. However, when FL-TFPI was complexed with heparin before addition to the plasma, the effect on anticoagulant activity was dependent on the weight ratio of heparin:FL-TFPI. Addition of the hepa rin:FL-TFPI complex at weight ratios <1.25:1 gave a dPT clot ting time shorter than that of addition of FL-TFPI alone sug gesting that neutralization of the positively charged carboxyl terminus of FL-TFPI by heparin could also decrease its anti coagulant activity. Addition of heparin:FL-TFPI complex at weight ratios ≥1.25:1 gave an additive or synergistic antico agulant effect compared to the individual anticoagulant effects of heparin and FL-TFPI. In contrast, addition of preformed N-acetyl heparin:FL-TFPI and low molecular weight hepa rin :FL-TFPI complexes in above weight ratios to plasma caused only inhibition of the anticoagulant activity of FL-TFPI. Pharmacokinetic studies of FL-TFPI and heparin:FL-TFPI complex were carried out in rabbits. Both pharmacokinetic data could be fitted into a biexponential clearance model. Full- length TFPI had a very short t1/2α (1.4 min) and a relatively long t1/2β (92 min) with AUCβ (92%) dominant. Heparin :FL- TFPI, in contrast, had a prolonged t1/2α (15 min) and a similar t1/2β (116 min) with AUCα (88%) dominant. The overall clear ance was ∼2.6-fold faster for FL-TFPI than heparin:FL-TFPI complex. Constant infusion studies confirmed that it was pos sible to achieve the same steady state level of TFPI in the circulating plasma by infusing 2.6-fold less complex than in fusion of the FL-TFPI alone.