Conversion of low-affinity platelet factor 4 to beta-thromboglobulin by plasmin and trypsin

Low-affinity platelet factor 4 and beta-thromboglobulin are platelet-secreted proteins that bind with low affinity to heparin. They show extensive immunological cross-reactivity and appear to differ in amino acid sequence only by an amino-terminal peptide unique to low-affinity platelet factor 4. The possibility that beta-thromboglobulin is derived from low-affinity platelet factor 4 by proteolysis was investigated by exposing this protein to the action of plasmin, thrombin and trypsin. While thrombin had no effect, plasmin and trypsin converted low-affinity platelet factor 4 to a species with the same electrophoretic mobility and isoelectric point as beta-thromboglobulin. We conclude that beta-thromboglobulin is a breakdown product of low-affinity platelet factor 4.     

Isolation, characterization, and immunological detection of neutrophil-activating peptide 2: a proteolytic degradation product of platelet basic protein.

Neutrophil-activating peptide 2 (NAP-2), corresponding to platelet basic protein fragment 25-94, was prepared by chymotryptic digestion of its precursors, low affinity platelet factor 4 or beta-thromboglobulin, followed by purification by high performance liquid chromatography. NAP-2 (0.1-1.5 microns) caused the release of human granulocyte elastase from cytochalasin B-treated neutrophils in a dose-dependent manner. In the same system, beta-thromboglobulin, human platelet factor 4, S-pyridylethyl NAP-2, and platelet basic protein C-terminal fragment (77-94) were inactive, whereas platelet basic protein fragment 22-89 had low, but significant, activity. Sensitive immunological identification of NAP-2 based on nonequilibrium isoelectric focusing and immunoblotting is described.     

Isolation and amino acid sequence of bovine platelet factor 4

Bovine platelet factor 4 was isolated by affinity chromatography using dextran sulfate Sepharose and purified by subsequent gel filtration. The complete amino acid sequence of this 88-residue, 9505-Da protein was determined by isolation and analysis of the overlapping peptides from tryptic and Staphylococcus aureus hydrolysates of reduced, carboxymethylated, and reductive methylated protein. Primary structure comparison was made between bovine platelet factor 4, human platelet factor 4, and human beta-thromboglobulin. The bovine platelet factor 4 amino-terminal region, which contains two unique phenylalanine residues, is extended by 15 residues relative to human platelet factor 4. The bovine carboxy-terminal region is extended by three residues relative to human platelet factor 4 and differed from beta-thromboglobulin in the absence of two additional terminal residues. Bovine platelet factor 4 shares sequence similarities proportionately with both human platelet factor 4 and beta-thromboglobulin. The sequences of the lysine-rich carboxy-terminal putative heparin binding domains are essentially identical for all three proteins. The heparin neutralizing potencies of bovine and human platelet factor 4 are similar: 40 USP units of heparin neutralized per milligram protein, as measured by a modified chromogenic substrate assay. Heparin neutralization was lost by reduction of the disulfide bonds, but only attenuated by tryptic digestion of the intact protein.     

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.     

Human platelet secreted proteins and prostacyclin production by bovine aortic endothelial cells

The effects of specific human platelet-secreted proteins on prostacyclin (PGI2) production by primary cultures of bovine aortic endothelial cells have been studied. Cells were incubated with various concentrations of highly purified preparations of platelet factor 4 (PF4), low-affinity platelet factor 4 (LA-PF4), beta-thromboglobulin (beta TG), platelet basic protein (PBP), and partially purified platelet-derived growth factor (PDGF) in the presence or absence of arachidonic acid (AA). The amount of 6-Keto-PGF1 alpha, the stable degradation product of PGI2, was determined in the cell incubation medium by means of a specific radioimmunoassay. Short-term (15 min) incubation of cell monolayers with either LA-PF4 or beta TG slightly reduced 6-keto-PGF1 alpha production. The effect was not dose-related and could not be observed after prolonged (24 hr) incubation of the cells with the same proteins. It was not seen in the cell suspensions. Moreover, 6-keto-PGF1 alpha production stimulated by AA was not affected by incubation with either of the proteins. PF4 and PBP had no significant effect on 6-keto-PGF1 alpha production by endothelial cells. Human PDGF showed a slight tendency to stimulate 6-keto-PGF1 alpha release when cells were incubated for 24 hr with the protein; however, PDGF did not potentiate the stimulatory effect of AA on 6-keto-PGF1 alpha release by the cells. We suggest that platelet-derived proteins exert only a moderate and possibly nonspecific effect on PGI2 production by endothelial cells.     

A collagen-binding glycoprotein from bovine platelets is identical to propolypeptide of von Willebrand factor

Several proteins from bovine platelet lysate bound to type I collagen immobilized to the beads of formyl derivatives of cellulose. Among these proteins, a protein of about 100,000 daltons was purified to homogeneity by two additional affinity chromatographies, an organomercurial-agarose and a lentil lectin-agarose. This protein consisted of a single polypeptide chain which contains carbohydrate moiety and many intrapolypeptide disulfide bridges. In addition to platelets, this protein was present in plasma and cultured endothelial cells but not in red blood cells, leukocytes, and smooth muscle cells. Furthermore, it was released from platelets upon stimulation by various agonists. The purified 100-kDa protein was labeled with 125I to quantitate its binding to fibrillar type I collagen. The protein specifically bound to fibrillar collagen with the apparent dissociation constant of 5.6 x 10(-8) M for the high affinity site and 5.5 x 10(-7) M for the low affinity site. Analyses of amino acid sequences of both intact and tryptic fragments of this protein revealed that it had strong homology to the propolypeptide of human von Willebrand factor, which is also known as von Willebrand antigen II. Various properties of this protein listed above also strongly suggest that it was indeed the propolypeptide of bovine von Willebrand factor.     

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent

1. A new method is described for labelling proteins to high specific radioactivities with (125)I. The protein is treated with a (125)I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the (125)I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55muCi/mug respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of (125)I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single (125)I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.     

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with ¹²⁵I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When ¹²⁵I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the ¹²⁵I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with ¹²⁵I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the ‘line’ form of platelet factor 4. This confirms that stable complexes of ¹²⁵I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.     

Interaction of platelet factor 4 with human platelets

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.     

Chemotactic activity of platelet alpha granule proteins for fibroblasts

At sites of blood vessel injury, platelets release numerous substances that may have biological activities influencing cellular responses. In this study we examined separately the chemotactic activity for fibroblasts of three highly purified proteins obtained from platelet alpha granules: platelet factor 4 (PF4), platelet-derived growth factor (PDGF), and beta-thromboglobulin (BTG). We observed that each of these proteins was strongly chemotactic for fibroblasts, with maximum chemotactic activity in each instance comparable to that observed with an optimal concentration of the control chemotactic protein, plasma fibronectin. Each protein was active at very low concentrations. The peak chemotactic activities of PF4, PDGF, and BTG occurred at 200 mg/ml, 30 ng/ml, and 6 ng/ml, respectively. Specificity of fibroblast chemotaxis to individual platelet proteins was provided by finding that anti-PF4 immunoglobulin blocked the chemotactic activity of PF4 without affecting the chemotactic activity of PDGF, while anti-PDGF immunoglobulin blocked the activity of PDGF but did not alter the capacity of PF4 to promote fibroblast chemotaxis. These results suggest that in vivo several alpha granule proteins released from platelets may affect wound healing by causing directed fibroblast migration.     

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Application to the radioimmunoassay

1. A new method is described for labelling proteins to high specific radioactivities with ¹²⁵I. The protein is treated with a ¹²⁵I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the ¹²⁵I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55μCi/μg respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of ¹²⁵I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single ¹²⁵I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.     

Human platelets possess receptors for a lymphokine: demonstration of high specific receptors for HuIFN-gamma

This report demonstrates that 125I-recombinant human interferon-gamma (125I-rHuIFN-gamma) binds to high-affinity specific receptors on human platelets. Scatchard analysis of binding data indicates the presence of homogeneous sites estimated in the order of 150 to 200, with an apparent equilibrium dissociation constant, Kd, of 2 X 10(-10) M. The binding of 125I-rHuIFN-gamma to platelet membrane was inhibited by unlabeled rHuIFN-gamma but not by unlabeled rHuIFN-alpha or unlabeled rHuIFN-beta. High affinity binding sites for HuIFN-alpha were not detectable. Cross-linking of 125I-rHuIFN-gamma to platelet membrane proteins with the use of a bifunctional agent (DSS) yielded a predominant complex of 100,000 +/- 5,000 daltons on SDS-PAGE autoradiography, which confirms the presence of specific receptors for IFN-gamma. Two faint bands of lower m.w., 70,000 and 90,000, could also be visualized. Cross-linking of 125I-rHuIFN-alpha to platelet surface could not be demonstrated by using the same procedures. This is the first time that a receptor for a lymphokine (IFN-gamma) has been demonstrated on human platelets. These findings are consistent with data already published, suggesting an interrelationship between IFN and platelet function.     

Complete covalent structure of human beta-thromboglobulin.

The complete primary structure of the platelet-specific protein human beta-thromboglobulin has been determined. beta-Thromboglobulin consists of identical subunits of 81 amino acids, each with a molecular weight of 8851. The amino acid sequence of the beta-thromboglobulin subunit is: Gly-Lys-Glu-Glu-Ser-Leu-Asp-Ser-Asp-Leu-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr-Thr-Ser-Gly-Ile-His-Pro-Lys-Asn-Ile-Gln-Ser-Leu-Glu-Val-Ile-Gly-Lys-Gly-Thr-His-Cys-Asn-Gln-Val-Glu-Val-Ile-Ala-Thr-Leu-Lys-Asp-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Pro-Asp-Ala-Pro-Arg-Ile-Lys-Lys-Ile-Val-Gln-Lys-Lys-Leu-Ala-Gly-Asp-Glu-Ser-Ala-Asp. Disulfide bridge-18 to half-cystine-58. The amino acid sequence of beta-thromboglobulin shows a marked homology with that of platelet factor 4. When the sequences are aligned for maximum homology, 42 of the 81 residues of beta-thromboglobulin are identical with those of platelet factor 4, including the position of the four half-cystines.     

The binding of beta-2-microglobulin to renal brush-border membrane: affinity measurement, inhibition by serum albumin

In the kidney, filtered proteins are rapidly reabsorbed so that the final excretion is less than 0.1% of the filtered amount for low molecular weight proteins such as beta 2-microglobulin and a few percent for albumin. In order to investigate the affinity of proteins for luminal membranes, rat renal brush-border membranes were incubated with 125I-labelled human beta 2-microglobulin and the initial binding rate determined by the filtration method. Scatchard plot analysis of binding rate revealed two types of binding sites: one with Km = 0.25.10(-6) M and Vmax = 0.1 nmol/min per mg protein and another with Km = 1.10(-5) M and Vmax = 1.3 nmol/min per mg protein. The lower affinity type is likely to represent non-specific binding the physiological role of which is to be discussed. The higher affinity sites seem to play the major role in binding rate. beta 2-Microglobulin initial binding is reversible, and inhibited by bovine serum albumin. Comparison of the time course of bound beta 2-microglobulin removal by unlabelled beta 2-microglobulin and by albumin suggests that these two proteins have a different internalization mechanism.     

Properties of a prolactin receptor from the rabbit mammary gland

Receptors for human, simian, ovine, bovine and murine prolactin, human growth hormone and human placental lactogen have been identified in plasma-membrane-containing subcellular particles isolated from rabbit mammary glands. The association and dissociation of (125)I-labelled prolactin are time- and temperature-dependent processes, both being maximal at 37 degrees C. (125)I-labelled prolactin prepared by the enzymic iodination procedure with lactoperoxidase binds better to receptors than does the preparation obtained by using chloramine-t as the oxidizing agent. The binding of (125)I-labelled prolactin to receptors is strongly influenced by pH and ionic composition but not by many low-molecular-weight compounds tested, e.g. steroids, nucleotides and several drugs. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipid moieties are essential for the binding of (125)I-labelled prolactin. The binding of (125)I-labelled prolactin to receptors is a saturable and reversible process. Scatchard and Lineweaver-Burk analyses suggest that (125)I-labelled prolactin has a high affinity for its receptor. Binding of (125)I-labelled prolactin to receptors does not result in the destruction of the hormone. Considerable prolactin-binding activity is also observed in subcellular fractions isolated from the adrenal gland, liver, ovary and kidney of the pregnant rabbit, a finding that is consistent with other reported actions of prolactin in these organs.     

Isolation of bovine platelet cationic proteins which inhibit the surface-mediated activation of factor XII and prekallikrein

A possible role of bovine platelets in the surface-mediated activation of Factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine plasma. The washed platelets before and after aggregation by ADP, thrombin or collagen did not show any ability to trigger or accelerate the activation of Factor XII and prekallikrein. On the contrary, these aggregates showed a potent inhibitory activity on the activation of those zymogens triggered by kaolin, amylose sulfate and sulfatide. The inhibitory substances from the supernatant of the thrombin-induced aggregates were separated into two major fractions, a low affinity fraction and a high affinity fraction, on a heparin-Sepharose column. The high affinity protein was identified as platelet factor 4, based on the amino acid composition. From the low affinity fraction, a beta-thromboglobulin (beta-TG)-like substance and three kinds of unknown proteins, named LA1, LA2, and LA3, were isolated by gel-filtration on a column of Sephadex G-100 or Sephadex G-75 followed by chromatography on a column of Mono S. The molecular weights of LA1, LA2, and LA3 were estimated to be 35,000, 26,000, and 11,000, respectively, on SDS-PAGE. LA2 was identified as a carbohydrate-less LA1, as judged from the amino acid composition and carbohydrate content. The inhibitory activities of these five cationic proteins on the activation of Factor XII and prekallikrein mediated with amylose sulfate, sulfatide and kaolin were different from each other. In the case of kaolin-mediated activation, LA3 was the most potent inhibitor, while platelet factor 4 and beta-TG-like substance did not show any significant inhibitory activity. Moreover, the inhibitory activities of all the cationic proteins were not correlated with their anti-heparin activities. Since these proteins were rapidly liberated from platelets by the action of the stimulants, the present results demonstrate a negative role of platelets in the surface-mediated activation of Factor XII and prekallikrein.     

Inhibition of epidermal growth factor binding to Swiss 3T3 cells by human platelet release-products

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.     

Characterization of somatostatin binding sites in cytosolic fraction of rat intestinal mucosa

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rat intestinal mucosa by using 125I-labelled Tyr11-somatostatin and a variety of physicochemical conditions. The binding depended on time, temperature and pH, and was reversible, saturable and specific. At apparent equilibrium, the specific binding of 125I-Tyr11-somatostatin was competitively inhibited by native somatostatin in the 1 nM-4 microM concentration range. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 0.07 microM) and low capacity (4.6 pmol/mg protein) and a class with low affinity (Kd = 1.05 microM) and high capacity (277 pmol/mg protein) at 25 degrees C. Somatostatin exhibited competitive inhibition of tracer binding, while neuropeptides such as neurotensin, substance P, Leu-enkephalin, and vasoactive intestinal peptide were ineffective. The presence of somatostatin binding sites in cytosolic fraction of intestinal mucosa, together with the known occurrence of somatostatin in D-cells and nerve endings in the small intestine, strongly suggest that this peptide may be involved in the physiology and physiopathology of intestinal epithelium.     

Calmodulin-binding proteins from brain and other tissues.

The calmodulin contents of rabbit brain, lung, kidney and liver, of bovine aorta and uterus, and of chicken gizzard have been determined. 2. The calmodulin in all of these tissues has been shown to be present in the form of very stable complexes with several other proteins. 3. A calmodulin-binding protein of mol.wt. 22 000 has been purified in high yield from bovine brain. It has been shown to interact with calmodulin and rabbit skeletal-muscle troponin C in a Ca2+-dependent manner. 4. The 22 000-mol.wt. protein inhibits the activation of bovine brain phosphodiesterase by calmodulin, but has very little affect on the activation of myosin light-chain kinase. 5. Calmodulin-binding proteins of mol.wts. 140000, 77000 and 61000 have also been partially purified from rabbit brain by affinity chromatography and have been shown to interact in a Ca2+-dependent manner with calmodulin. 6. The apparent molecular weights of the calmodulin-calmodulin-binding protein complexes, determined by gel filtration in the presence of 6M-urea, have been shown to be similar for most of the mammalian tissues examined. 7. By using 125I-labelled calmodulin, similar complexes have been demonstrated in rabbit skeletal muscle, although they are present at much lower concentrations.