Protease nexin-II (PN-II) [amyloid beta-protein precursor (APP)] and the amyloid beta-protein are major constituents of neuritic plaques and cerebrovascular deposits in individuals with Alzheimer's disease and Down syndrome. Both the brain and the circulation have been implicated as sources of these molecules, although they have not been detected in blood. Human platelets have now been found to contain relatively large amounts of PN-II/APP. Platelet PN-II/APP was localized in platelet alpha-granules and was secreted upon platelet activation. Because PN-II/APP is a potent protease inhibitor and possesses growth factor activity, these results implicate PN-II/APP in wound repair. In certain disease states, alterations in platelet release and processing and clearance of PN-II/APP and its derived fragments could lead to pathological accumulation of these proteins.

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.

The vasoactive compound bradykinin (BK) is liberated by proteolytic cleavage from high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Expression of kininogens on cell surface receptors may affect the delivery of BK at sites of inflammation. Therefore, we investigated whether BK itself alters the expression of binding sites for its parent molecules, HK and LK, on the surface of cultured human umbilical vein endothelial cells (HUVEC). 125I-LK and 125I-HK each bind to a single class of sites on HUVEC in reactions that are saturable, reversible, and zinc-dependent (Bmax = 9.7 +/- 0.2 x 10(5) sites/cell; kd = 43.3 +/- 8 nmol/L; n = 5 and Bmax = 10.3 +/- 0.4 x 10(5) sites/cell; kd = 40.3 +/- 0.9 nmol/L; n = 3 for LK and HK, respectively). HK and LK compete for the same binding site (Ki = 19.4 +/- 5 nmol/L HK v 125I-LK; Ki = 24.5 +/- 4 nmol/L LK v 125I-HK, n = 3). Moreover, 50-fold molar excess light chain of HK inhibits 125I-LK binding 51% and 50-fold molar excess LK and the heavy chain of HK inhibit 125I-light chain of HK binding 92% and 76%, respectively. Preincubation of HUVEC with BK produces a transient, concentration- dependent increase in the binding of HK and LK, reaching a maximum 3 to 4 hours after addition of BK (46% increase over control for HK; 57% increase over control for LK; P < .005 for each ligand). Des-Arg9- bradykinin, a B1 receptor agonist, increases kininogen binding to the same extent as BK; the upregulation of kininogen binding sites by BK is partially blocked by a B1 but not by a B2 receptor antagonist. The protein kinase C inhibitors (PKC), sphingosine and H7, completely block the induction of HK receptors by BK. Phorbol 12-myristate 13-acetate (PMA), which also activates PKC, stimulates the binding of HK and the purified light chain of HK to HUVEC as well. However, unlike HK and its light chain, binding of LK and the heavy chain of HK are increased by PMA only in the presence of added calcium ion. These studies show that BK upregulates a common binding site for HK, LK, and each chain of HK on HUVEC. Induction of kininogen receptors on endothelial cells by BK may modulate the generation of this vasoactive compound at sites of vascular injury.

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

Using the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCl, an amidolytic assay was designed to measure prekallikrein in plasma. At a substrate concentration of 1 mM (Km = 0.2 mM), the amidolysis of purified kallikrein at 1 coagulant unit/ml was observed to be 2.47 mumole/min/ml. Conditions for plasma prekallikrein activation were optimized to approach complete activation when compared to the amidolytic activity of the purified plasma kallikrein. Plasma treated with chloroform to destroy inhibitors of kallikrein was activated with dilute kaolin (final concentration 1 mg/ml) for 1 min at 25 degrees C. Activated plasma prekallikrein had 78% (1.92 mumole/min/ml) of activity of purified kallikrein at plasma concentration. Comparison of this amidolytic assay with immunochemical, esterolytic, and coagulant assays of three subject populations (normals, women on birth control pills, and patients with hepatocellular disease) showed good correlation both in normals and in the patient groups between the amidolytic and esterolytic assays (r = 0.89). Each enzymatic assay correlated with the immunochemical assay (r = 0.72, r = 0.68, respectively). However, comparison of each of these assays with the coagulant assay showed no significant correlation due to the large inherent error of the latter assay. This standardized plasma prekallikrein amidolytic assay should facilitate studies of plasma prekallikrein concentration in physiologic and pathologic conditions and help identify activation of the contact phase of coagulation in disease states.