Human plasma contains a factor Xadependent inhibitor of tissue factor/factor VI~a complex termed lipoprotein-associated coagulation inhibitor (LACI). The present study examines the site(s) of LACI synthesis. In this study, cultured hepatocytes isolated from normal human liver were found to be essentially negative in LACI mRNA as revealed by Northern blot analysis using a full-length LACI cDNA as probe. The conditioned media from these cultures were also essentially negative for LACI activity. Similarly, poly(A)+ RNA obtained from normal human liver did not contain detectable LACI mRNA. In contrast, cultured human umbilical vein endothelial cells and human lung tissue (rich in endothelium) both contained abundant amounts of LACI mRNA. Moreover, erythrocyte lysates and culture media from normal monocytes, lymphocytes, or neutrophils did not contain measurable LACI activity; these cells were also negative for LACI mRNA. Platelets, however, contained LACI activity. The likely source of platelet LACI is the megakaryocyte cell since a megakaryocyte cell line (MEG-01) was found to contain LACI mRNA and to secrete small amounts of LACI activity.Additionally, human vascular smooth muscle cells and lung fibroblasts were also found to synthesize only small amounts of LACI. From these observations, we conclude that normal liver does not synthesize LACI and that endothelium is the principal source of plasma LACI. The undegraded LACI synthesized by endothelial cells had a molecular weight of 41,000.

A simplified procedure is described for the purification of prothrombin, Factor X and Factor IX in overall yields of 35-40% from pooled human plasma. The initial steps, which are common to prior purification techniques, include adsorption onto and elution from barium citrate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. The procedure differs from previous techniques in that the nest step, heparin-agarose chromatography, is carried out in a (sodium) citrate buffer, pH 7.5. These chromatographic conditions permit the separation of prothrombin, Factor X and Factor IX from each other, yielding fractions with apparent homogeneity in several electrophoretic systems. The additional chromatographic steps of earlier purification procedures are therefore unnecessary. The heaprin-agrarose column chromatographic conditions consistently resulted in the separation of human prothrombin in into two fractions in a ratio of approximately 4:1. Both fractions possess similar specific activity in a one stage prothrombin assay, and also activate at the same rate in a Factor Xa, Ca2+ and phospholipid system. Both fractions of prothrombin also comigrate in sodium dodecyl sulfate gel electrophoresis with an apparent Mr integral of 70,000.

SummarySpecific antibodies to tissue factor pathway inhibitor (TFPI) were used in immunohistochemical procedures to determine the distribution of TFPI in normal and neoplastic human tissues. TFPI was restricted to megakaryocytes and the endothelium of the microvasculature in normal and abnormal tissues, but was not found in the endothelium of larger vessels or in hepatocytes. TFPI was also detected in macrophages in the villi of term placenta. Tumor-associated macrophages in several types of malignancy that we have shown previously to express a complete tissue factor-initiated pathway of coagulation and thrombin generation also manifested TFPI. By contrast, malignant cells in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma that we have shown previously to express coagulation factors together with tumor cell-associated fibrin formation failed to stain for TFPI. We postulate that TFPI may be lacking from the latter malignancies because of the absence of the appropriately configured tissue factor – factor VII a – factor Xa complex required for TFPI binding.

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Fulllength TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2 position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2 residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ϳ6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ϳ8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.

Antibodies that bind prothrombin without neutralizing its coagulant activity were demonstrated in the plasma of two patients with the acquired hypoprothrombinemia-lupus anticoagulant syndrome. The first patient's plasma contained less than 1% prothrombin activity and no detectable prothrombin antigen. The second patient's plasma contained about 6% of both prothrombin activity and antigen. Neither patient's plasma neutralized the prothrombin coagulant activity of normal plasma or of purified prothrombin added in vitro. Nevertheless, double immunodiffusion studies and binding experiments utilizing 125I- prothrombin demonstrated the presence of prothrombin antibodies in each patient's plasma. A Scatchard analysis of the binding data obtained with different concentrations of 125I-prothrombin and the first patient's plasma indicated the presence of a high affinity antibody, apparent Kd approximately 10(-10)M, and a lower affinity antibody, apparent Kd approximately 10(-9)M. Studies utilizing purified cleavage products of prothrombin suggested that the antibodies were directed against an epitope or epitopes located on the carboxyl-terminal, latent thrombin segment of the prothrombin molecule. We postulate that the acquired hypoprothrombinemia in these two patients and in other reported patients with the acquired hypoprothrombinemia-lupus anticoagulant syndrome stems from rapid clearance from the circulation of prothrombin antigen-antibody complexes.