Human endothelial cells in culture and in vivo express on their surface all four components of the glycoprotein Ib/IX/V complex.

The platelet glycoprotein Ib (GpIb) complex is composed of four polypeptides: the disulfide-linked GpIb alpha and GpIb beta and the noncovalently associated GpIX and GpV. GpIb alpha contains binding sites for von Willebrand factor and for thrombin and mediates platelet adhesion to the subendothelium under conditions of high shear stress. We have previously shown the presence of GpIb alpha and GpIb beta mRNA and protein in cultured human umbilical vein endothelial cells (HUVECs) as well as the presence of GpIb alpha mRNA and protein in tonsillar endothelium. We, therefore, probed ECs for the presence of the other components of the GpIb/IX/V complex. We have identified the presence of GpIX and GpV mRNA in cultured HUVEC monolayers. The sequence of HUVEC GpIX cDNA was identical to the previously published human erythroleukemia (HEL) cell GpIX cDNA sequence. Two species of GpV mRNA, one of 3 kb and one of 4.4 kb, were found in HUVECs, whereas HEL cells displayed only the 4.4-kb species and the megakaryocytic cell line CHRF-288 contained only the 3-kb species. We previously showed that EC GpIb alpha protein is identical in molecular weight to platelet GpIb alpha. HUVEC GpIb beta, in contrast to its platelet counterpart, has a molecular weight of 50 kD and forms a correspondingly larger disulfide-bonded complex with EC GpIb alpha. The molecular weights of GpIX and GpV were 22 and 88 kD, respectively, identical to the corresponding platelet polypeptides. Furthermore, we have identified all four components of the complex in tonsillar vessels. Using flow cytometry, we have established that all four polypeptides of the GpIb/IX/V complex are expressed on the surface membranes of cultured HUVECs and adult aortic ECs. Furthermore, using two-color fluorescence, we have shown that all ECs expressing GpIb alpha also express GpIX and GpV on their surface. The ratio of GpIb alpha:GpIX:GpV is 1:1:0.5, which is identical to the ratio present in platelets. None of the polypeptides of the GpIb complex could be identified on the surface of human smooth muscle cells or lymphocytes. The presence of all members of the GpIb complex in the EC membrane suggests that this complex may play a role in endothelial function in vivo.

Platelet glycoprotein (Gp) IX associates with GpIb alpha in the platelet membrane GpIb complex.

The platelet membrane glycoprotein (Gp) Ib complex consists of four polypeptides: the disulfide-linked GpIb alpha and GpIb beta subunits; GpIX, tightly, but noncovalently associated with GpIb alpha-beta; and the more weakly associated GpV. It is not certain whether the association of GpIX to Gplb alpha-beta is via GpIB alpha, GpIb beta, or both subunits, although recently published evidence implicates an interaction with GpIb beta. We have investigated the interaction of GpIX with GpIb alpha-beta using polyclonal rabbit antibodies to GpIb alpha and GpIb beta raised by immunization with purified glycocalicin and with synthetic peptide sequences from GpIb beta, respectively, as well as monoclonal antibodies directed against GpIX (FMC-25) and against GpIb alpha (AP-1). We performed two types of experiments, using either purified GpIb complex or platelets. (1) When wells were coated with nonreduced GpIb complex, the binding of FMC-25 was inhibited 73% by GpIb alpha antibody, but only 30% by the GpIb beta antibody; when wells were coated with reduced complex, FMC-25 binding was inhibited by the same two antibodies by 86% and 13%, respectively. When wells were coated with polyclonal GpIb alpha or GpIb beta antibodies and then incubated with reduced GpIb complex, only wells coated with GpIb alpha antibodies captured GpIX reactivity. When wells were coated with FMC-25 and then incubated with nonreduced GpIb complex, both the GpIb alpha and GpIb beta polyclonal antibodies reacted strongly; in contrast, only GpIb alpha reactivity was retained when wells coated with FMC-25 were incubated with reduced GpIb complex. In the reciprocal experiment, AP-1-coated wells incubated with either nonreduced or reduced GpIb complex bound radiolabeled FMC-25. (2) The ability of polyclonal GpIb alpha and GpIb beta antibodies to inhibit binding of FMC-25 to platelets was studied by ELISA and by flow cytometry. In both systems, FMC-25 binding was inhibited by the GpIb alpha antibody, but not significantly by the GpIb beta antibody. We conclude that GpIX is strongly associated with GpIb alpha in the purified platelet GpIb complex and in the platelet membrane.

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

Glycoprotein Ib beta (GPIb beta) exists in platelets disulfide-linked to glycoprotein Ib alpha (GPIb alpha), a major receptor for von Willebrand factor. Both GPIb alpha and GPIb beta are expressed in endothelial cells (EC). While the GPIb alpha mRNA and protein appear similar in platelets and EC, EC GPIb beta mRNA is larger than platelet GPIb beta and encodes a larger protein. We have cloned and sequenced EC GPIb beta cDNA and report a 2793-nucleotide sequence which contains a 411-amino acid open reading frame. The EC sequence contains all of the platelet cDNA sequence and all but three amino acids of the primary translation product. Like the genes encoding GPIb alpha, GPIX, and GPV, the GPIb beta gene appears simple in structure. Using human hamster hybrids, we have localized the GPIb beta gene to chromosome 22pter-->22q11.2. When we examined poly (A)+ RNA from several human tissues for GPIb beta mRNA expression, we found that GPIb beta mRNA was expressed in a variety of tissues but was most abundant in heart and brain, while GPIb alpha and GPIX mRNA expression was found only in lung and placenta at very low levels. The broad distribution of GPIb beta mRNA suggests that it may be playing a role different than or additional to its function in platelets.

PPACK-thrombin is a noncompetitive inhibitor of alpha-thrombin binding to human platelets.

Recent studies from our laboratory indicate that purified kininogens are noncompetitive inhibitors of human alpha-thrombin but not PPACK-thrombin, binding to human washed platelets. In order to understand the mechanism by which the kininogens inhibit alpha-thrombin binding, investigations were initiated to determine if alpha-thrombin and PPACK-thrombin bound to the same site on human platelets. Initial investigations reveal that alpha-thrombin is a more potent inhibitor of 125I-PPACK-thrombin binding than PPACK-thrombin. Further studies show that PPACK-thrombin is a noncompetitive inhibitor of 125I-alpha-thrombin binding to platelets. These studies suggest that human alpha-thrombin binds on the platelet surface to a different site or binds differently to the same site from PPACK-thrombin. These data indicate that the ability of the kininogens to block alpha-thrombin binding to platelets but not PPACK-thrombin binding results from these thrombins having either two different binding sites or one binding site on the platelet surface which they interact with differently.

Insights on monoclonal antibodies to kininogens' heavy chain which influence kininogens' binding to platelets.

Purified domains of low molecular weight kininogen (LK) can be used directly to determine the epitopes of monoclonal antibodies (mAbs) that have been shown to influence kininogen function. LK, purified from plasma by carboxymethyl-papain-Sepharose 4B affinity chromatography and kaolin adsorption, was digested by trypsin and chymotrypsin. The domains of LK were then separated by gel filtration followed by carboxymethyl-papain-Sepharose 4B affinity chromatography. Using the purified domains of LK's heavy chain, the regions on kininogens' heavy chain which various monoclonal antibodies are directed to were determined by enzyme-linked immunosorbent assay and immunoblotting. MAb 2B5 which neutralized kininogens' ability to inhibit calpain cross-reacted with domains 2 and 3. MAb HKH8 which reacted with kininogens' domain 1 and 2 was found to inhibit 125I-HK binding to platelets. At two-fold molar excess, mAb HKH8 was a better inhibitor of 125I-HK binding to platelets than higher concentrations, where the antibody was shown to cause increased binding to platelets. Alternatively, HKH8 F(ab')2 completely inhibited 125I-HK binding to platelets even at high concentrations of antibody. These studies indicate that purified domains of kininogens' heavy chain can be used to rapidly localize epitopes for antibodies. Further, mAb HKH8 should be a valuable probe to understand the mechanisms of kininogens' binding to platelets.

High molecular weight kininogen binds to platelets by its heavy and light chains and when bound has altered susceptibility to kallikrein cleavage.

The unstimulated platelet surface contains a specific and saturable binding site for high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Investigations were performed with purified heavy and light chains of HK to determine which portion(s) of the HK molecule binds to the platelet surface. Purified 64-Kd heavy chain of HK and 56-Kd light chain of HK, independently, inhibited 125I-HK binding to unstimulated platelets with a 50% inhibitory concentration (IC50) of 84 nmol/L (apparent Ki, 30 nmol/L) and 30 nmol/L (apparent Ki, 11 nM), respectively. The ability of each of the purified chains of HK to independently inhibit 125I-HK binding was not due to cleavage, reduction, and alkylation of the protein, because two-chain HK, produced by treating HK the same way as purifying the separate chains, inhibited binding similarly to intact HK. Further, purified LK alone inhibited 125I-HK binding to platelets (Ki, 17 +/- 1 nmol/L, n = 7). The 64-Kd heavy chain of HK was a competitive inhibitor on a reciprocal plot of 125I-HK-platelet binding with an apparent Ki of 28 +/- 6 nmol/L (n = 4). Independently, purified 56-Kd light chain of HK was also found to be a competitive inhibitor of 125I-HK-platelet binding, with an apparent Ki of 11 +/- 3 nmol/L (mean +/- SEM, n = 4). These indirect studies indicated that HK binds to platelets by two portions of the molecule, one on the heavy chain and another on the light chain. Studies with 125I-light chain of HK showed that it specifically bound directly to platelets in the presence of zinc, since it was blocked by HK, light chain of HK, or EDTA, but not by LK, C1s, C1 inhibitor, plasmin, factor XIII, or fibrinogen. Purified light chain of HK did not inhibit direct 125I-LK binding to platelets. HK was found to bind to platelets in an unmodified form. HK bound to platelets was cleaved by plasma or urinary kallikrein at a slower rate than the same concentration of soluble HK or HK bound and subsequently eluted from the platelet surface. Cleavage of platelet-bound HK correlated with bradykinin liberation. These studies indicate that HK has two domains on its molecule that bind to platelets. Further, platelet-bound HK is protected from kallikreins' proteolysis. This latter finding suggests that cell binding may modify the rate of bradykinin liberation from HK.

The rate of fibrinopeptide B release modulates the rate of clot formation: a study with an acquired inhibitor to fibrinopeptide B release.

An asymptomatic 50-year-old male with a gamma globulin paraprotein was found to have prolonged prothrombin time, activated partial thromboplastin time, and thrombin time but a normal reptilase time. The prolonged clotting times were not the result of a factor deficiency because they were not corrected by the addition of normal plasma. Instead, this patient had an antibody that delayed thrombin-mediated fibrinopeptide B release thereby producing an apparent dysfibrinogenaemia. His isolated IgG prolonged the thrombin clotting time of both normal plasma and fibrinogen. Precincubation of his IgG with fibrinopeptide B, but not with fibrinopeptide A or thrombin, decreased its ability to prolong the thrombin clotting time. The patient's purified IgG but not control IgG delayed thrombin-mediated fibrinopeptide B release from fibrinogen without affecting the release of fibrinopeptide A. These studies define a novel, clinically silent dysfibrinogenaemia due to an antibody that delays thrombin-mediated fibrinopeptide B release from fibrinogen thereby markedly prolonging the clotting times.

Low molecular weight kininogen binds to platelets to modulate thrombin-induced platelet activation.

The kininogens, high molecular weight kininogen (HK) and low molecular weight kininogen (LK), are multifunctional, single-gene products that contain bradykinin and identical amino-terminal heavy chains. Studies were performed to determine if LK would bind directly to platelets. 125I-LK specifically bound to gel-filtered platelets in the presence of 50 microM Zn2+. HK effectively competed with 125I-LK for the same binding site (Ki = 27 +/- 9 nM, n = 5). Similarly, the Ki for LK inhibition of 125I-LK binding was 12 +/- 1 nM (n = 3). Albumin, fibrinogen, factor XIII, and kallikrein did not inhibit 125I-LK binding to unstimulated platelets. 125I-LK (66 kDa) was not cleaved upon binding to platelets. The binding of 125I-LK to unstimulated platelets was found to be fully reversible by the addition of a 50 molar excess of unlabeled LK at both 10 and 20 min. LK binding to platelets was saturable with an apparent Kd of 27 +/- 2 nM (mean +/- S.E., n = 9) and 647 +/- 147 binding sites/platelet. Both LK and HK at plasma concentrations inhibited thrombin-induced platelet aggregation. LK and HK at about 5% of plasma concentration also inhibited thrombin-induced secretion of both stirred and unstirred platelets. Both kininogens were found to be noncompetitive inhibitors of proteolytically active thrombin binding to platelets. The kininogens did not inhibit D-phenylalanyl-prolyl-arginine chloromethyl ketone-treated thrombin from binding to platelets. These studies indicated that both kininogens have a region on their heavy chain which allows them to bind to platelets. Further, kininogen binding by its heavy chain modulates thrombin activation of platelets since it prevents proteolytically active thrombin from binding to its receptor.

Immunoblotting of plasma in a pregnant patient with hereditary angioedema.

Hereditary angioedema (HAE), an autosomal disorder caused by a deficiency of C1 inhibitor, is characterized by attacks of localized swelling, laryngeal edema, or abdominal pain. Plasma samples from one pregnant patient were studied serially by functional and quantitative immunochemical assays as well as immunoblot assays for high molecular weight kininogen (HMWK) and/or prekallikrein/kallikrein (PK/K). An immunoblot of this patient's HMWK from plasma obtained before she became pregnant and when she was well revealed that it was mostly an intact protein of 120 kd, similar to immunoblot results of normal plasma HMWK. In plasma samples taken throughout her pregnancy, before, during, and after clinical attacks of angioedema, all of her plasma HMWK was shown to be cleaved into the 45 kd light chain form. After delivery of the infant the 120 kd form of intact plasma HMWK returned to her plasma. In comparison, immunoblot studies on 21 normal and abnormal pregnancies revealed that plasma HMWK was an intact protein at 120 kd. That this patient's plasma during her pregnancy was contact activated was determined by additional immunoblot studies for PK/K. Immunoblot assay for plasma PK/K revealed kallikrein-alpha 2-macroglobulin complexes and a 50 kd PK/K form seen only in activated plasma samples. The findings of kallikrein-alpha 2-macroglobulin complexes and a 50 kd PK/K form disappeared after delivery. These combined studies on this patient show that the structures of HMWK and prekallikrein as indicated by immunoblot assays were altered during pregnancy. Immunoblot assays for detection of changes in the structure of HMWK and prekallikrein may be objective laboratory studies for documenting clinical attacks of hereditary angioedema, their onset, and their resolution.