Proteins Platelet Factor Research Articles

Hollow Fiber Membrane Oxygenator Reduces Platelet Loss During Simulated Extracorporeal Circulation

Contact between blood and the extracorporeal circuit results in adverse alterations in platelet number and function. To determine the effects of surface composition and flow on these changes, 450 ml of heparinized (5 U/ml) blood from random, aspirin-free volunteers was recirculated at a flow rate of twice the circulating volume for 2 hrs at 37°C through circuits with an identical surface area (1.0m2). These circuits contained either a spiral coil, silicon rubber, membrane oxygenator (SC) or a hollow fiber, polypropylene membrane oxygenator (HF). In SC circuits (n=5), platelet counts fell to 10±3% (±SEM p <.05) at 5 min. rising to 60±9% (p < .05) at 2 hrs. of extracorporeal circulation (ECC). In contrast, in HF circuits (n=5), the platelet count fell to only 53±7% (p < .05) at 5 min of ECC and rose to 82 + 14% (p > .05) at 2 hrs. Plasma levels of the platelet-specific protein platelet factor (PF) 4 rose to 2780 ± 222 ng/ml (p <.05) by 5 minutes, reaching 6338 ±.767 ng/ml (p < .05) at 2 hours in SC circuits. In contrast, PF4 in HF circuits rose to 836±73 ng/ml and 2640 ± 410 ng/ml (p < .05), respectively. Although platelets in the SC circuits became insensitive to ADP ≥ 50 uM) within 5 min, platelets from HF circuits aggregated to ADP (17 uM) despite 2 hrs of ECC. We conclude that the hollow fiber membrane oxygenator not only reduces adhesion, maintaining the circulating platelet count, but also reduces platelet protein release and preserves platelet function as well.

Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [ G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the ArgGlyAspSer ( RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1μg/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating “receptors” for PGs that permit stable interaction. These data provide evidence that two receptors for the fibronectins, integrin and heparan sulfate proteoglycan, interact with cell-surface GM1 in a “ cis-oriented” manner to regulate adhesive responses of these neural cells to ganglioside-binding substrata. Two alternative mechanisms of this interaction are discussed.

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (antiCBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.

Possible interaction of platelets and adrenaline in the early phase of myocardial infarction

It is known that in most cases of transmural acute myocardial infarction a platelet clot originates within a coronary artery. In acute myocardial infarction patients increased levels of the plasma catecholamines adrenaline and noradrenaline as well as the platelet release proteins platelet factor 4 and beta-thromboglobulin have been reported. In this study, significantly higher values were found of platelet factor 4 (P less than 0.0001) and beta-thromboglobulin (P less than 0.002) in 17 acute myocardial infarction patients as compared to 17 control patients (on intensive care due to non-cardiac disorders), while the plasma levels of adrenaline and noradrenaline were not different. Positive correlations were obtained between the two catecholamines and the platelet products in the control group and between adrenaline and both platelet factor 4 (r = 0.715, P less than 0.01) and beta-thromboglobulin (r = 0.547, P less than 0.05) in the acute myocardial infarction patients. The data suggest that a stimulation of the platelets by adrenaline may facilitate in vitro activation during sampling in patients with high catecholamine load. On the other hand, a "preactivation" of the platelets by an increase of adrenaline might be of significance for thrombus formation in acute myocardial infarction.

Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3′-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5′ region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

Cloning and Characterization of Platelet Factor 4 cDNA Derived From a Human Erythroleukemic Cell Line

We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3'-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5' region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4.

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.

Acute postprandial lipaemia does not influence the [formula omitted] activity of human platelets

Platelet function and serum lipid studies were conducted on nine healthy male subjects before and two hours after three separate meals, two of which were rich in fat: saturated fat (SF) or polyunsaturated fat (PUF), and a third control meal which contained no fat. Platelet activity was assessed by determination of in vivo platelet aggregate formation, and plasma levels of the platelet specific proteins platelet factor 4 (PF 4) and β-thromboglobulin (β-TG). Serum triglyceride levels increased significantly after both fat containing meals but were unaffected by the fat free meal. Serum cholesterol levels were not affected by any of the three meals. Platelet function tests could not detect any alteration of in vivo platelet activity in terms of platelet aggregate formation or plasma concentrations of PF 4 and β-TG. These results are at variance with previously published studies that used less specific assays of platelet activity and which suggested platelet activation shortly after meals rich in SF.

Serial studies of platelet factor 4 and beta thromboglobulin during exercise in patients with coronary artery disease

In vivo activation of platelets can be accurately measured by radioimmunoassays of platelet factor 4 (PF4) and beta thromboglobulin (βTG). Studies that attempt to correlate increases in PF4 and βTG levels with exercise-induced myocardial ischemia have yielded conflicting results. To further examine the natural history of release of PF4 and βTG we used a method of serial samplings of these proteins during and after exercise in nine normal subjects and 24 patients with coronary artery disease (CAD). Mean values for PF4 and βTG at rest, during each stage, and immediately after treadmill exercise were the same for normal subjects and for patients with positive and negative responses to exercise-tolerance tests (ETTs). However, nonparametric analysis and regression equations disclosed differences in trends of PF4 level during exercise; PF4 levels increased in normal subjects during exercise, while patients with positive ETTs had no change in PF4 levels and patients with negative ETTs actually showed a decrease in PF4. This investigation confirmed that exercise-induced myocardial ischemia is not associated with platelet aggregation as manifested by the release of the platelet-specific proteins PF4 and βTG. Statistical analysis suggested that prior reports of elevated levels of PF4 during exercise could have been caused by technical and methodologic difficulties that were associated with the collection and handling of the samples.

Iloprost (ZK36374), a stable analogue of prostacyclin, preserves platelets during simulated extracorporeal circulation

Contact between blood and synthetic surfaces of an extracorporeal circuit results in extensive alterations in platelet function. These platelet abnormalities have been reproduced in vitro by recirculating heparinized (5 units/ml) human blood at 37 degrees C in a silicone rubber circuit (0.1 m2) containing a spiral coil membrane oxygenator (0.9 m2). During control recirculation trials, platelet counts fell to 29% +/- 8% (mean +/- standard error of the mean) of initial levels within 30 minutes, and sensitivity to the soluble agonists, adenosine diphosphate and epinephrine, disappeared. Plasma levels of the platelet-specific protein platelet factor 4 rose to 2,600 +/- 200 ng/ml, indicating extensive platelet granule release. Similarly, plasma concentrations of thromboxane B2 rose from less than or equal to 100 pg/ml to 500 +/- 200 pg/ml at 120 minutes, and transmission electron microscopy demonstrated disruption of platelet subcellular architecture. In contrast, when ilioprost, a stable prostacyclin derivative (1 ng/ml), was added to the circuit prior to recirculation, the thrombocyte count remained at 85% +/- 4% of initial values. Platelets incubated or recirculated for 2 hours in the presence of iloprost responded normally to both adenosine diphosphate and epinephrine after separation from the drug by gel-filtration. Furthermore, plasma concentrations of platelet factor 4 remained below 300 ng/ml, plasma levels of thromboxane B2 failed to reach detectable levels, and platelet ultrastructure remained intact. Thus, iloprost effectively preserves the circulating platelet count, prevents platelet granule release, and preserves platelet functional and morphological integrity during simulated extracorporeal circulation.

Platelet activation and secretion associated with emotional stress.

Platelets are believed to play a role in the pathogenesis of atherosclerosis and of the vascular obstruction that causes the acute complications of coronary artery disease. Since specific behavioral patterns appear to be related to the development of coronary artery disease and since emotional stress may predispose an individual to acute cardiovascular ischemia, it was hypothesized that platelet activation by catecholamines might be involved in these events. To study emotional stress, plasma samples were obtained from 61 senior medical residents immediately before they were to speak in public. There were significant increases in the plasma concentrations of the platelet-secreted proteins platelet factor 4 and beta-thromboglobulin and epinephrine and norepinephrine immediately before speaking, which demonstrates that platelet activation and secretion occur in association with this type of emotional stress. Four trials were carried out to study the mechanism for this observed platelet secretion: (1) phenoxybenzamine, (2) propranolol, (3) 650 mg aspirin, and (4) 80 mg aspirin were given several hours before the public speaking engagement. Neither phenoxybenzamine nor propranolol in doses that blocked the hemodynamic effects of alpha 1- and beta 1-adrenergic stimulation modified platelet secretion. Aspirin also did not block platelet secretion, which suggests that platelets were not being stimulated through a cyclooxygenase-dependent pathway. This study provides direct evidence of platelet secretion in vivo in association with emotional stress, and underscores the potential importance of platelet activation and secretion in the acute events that occur in patients with vascular disease.

Double blind study of the influence of co-dergocrine on platelet parameters in healthy volunteers

In 20 healthy volunteers double-blind randomized study was done to examine the effect of co-dergocrine (Hydergine) on platelet function. All the volunteers were pretreated with 3 X 1 placebo for 1 week, followed by the randomized study in which 10 volunteers each received drug or placebo for 6 weeks. Various platelet function parameters, such as the peripheral platelet count, plasma thromboxane B2, platelet sensitivity to the antiaggregatory prostaglandins PGI2, PGE1 and PGD2, ADP-induced aggregation, collagen-induced aggregation, the WU-test, the platelet proteins platelet factor 4 and beta-thromboglobulin, malondialdehyde, circulating endothelial cells and the intracellular c-AMP level in platelets were examined. Treatment with co-dergocrine decreased platelet activity to a significant extent, as shown by a number of platelet function parameters, such as thromboxane B2, WU-test, beta-thromboglobulin, platelet factor 4, malondialdehyde and ADP-induced aggregation. The findings suggest that co-dergocrine might be able to decrease platelet activity and improve interaction with the walls of to a blood-vessel significant degree.

Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. Affinity fractionation on columns of platelet factor 4, plasma fibronectin, and octyl-sepharose.

Both newly formed and long-term culture-generated substratum adhesion sites, generated by EGTA-mediated detachment of Balb/c SVT2 cells, were extracted with an eta-octyl-beta-D-glucopyranoside buffer containing salt and several protease inhibitors under conditions which result in maximal solubilization of the sulfate-radiolabeled proteoglycans. Because of the functional importance of heparan sulfate proteoglycans in the fibronectin-dependent cell-substratum adhesion processes of these cells, these proteoglycans were fractionated on affinity columns of octyl-Sepharose or of the heparan sulfate-binding proteins platelet factor 4 or plasma fibronectin. These affinity matrices resolved a number of both binding and nonbinding classes of heparan sulfate proteoglycan from both types of adhesion sites. In particular, the platelet factor 4 column could resolve several proteoglycans with differing binding affinities. Approximately twice as much heparan sulfate proteoglycan from newly formed sites bound to all three matrices as proteoglycan from longterm sites. The proteoglycan which bound to one matrix was then tested for binding to a second matrix; this approach resolved a number of biochemically distinct species. For example, one-half of the fibronectin-Sepharose-binding fraction from the long-term sites could also bind to platelet factor 4-Sepharose; however, over 90% of the fibronectin-binding fraction from newly formed sites could bind to platelet factor 4. A major portion of the octyl-Sepharose-binding fractions of the original extracts could bind to fibronectin-Sepharose. These studies indicate that some of these proteoglycans have overlapping affinities for fibronectin, platelet factor 4, and octyl-Sepharose and that a portion of the heparan sulfate proteoglycan from these adhesion sites cannot bind to any of these affinity matrices. These results are discussed with regard to the functional significance of these various heparan sulfate proteoglycans in mediating adhesion to extracellular matrices containing fibronectin or platelet factor 4.

Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglobulin in thin frozen sections of human blood platelets.

Affinity-purified monospecific antibodies against human fibrinogen and the platelet-specific proteins platelet factor 4 and beta thromboglobulin were used to localize these antigens in thin and ultra-thin frozen sections of mildly fixed, washed human blood platelets. By immunofluorescent double-labeling experiments the distribution of fibrinogen was compared to that of platelet factor 4 and beta thromboglobulin. All three antigens occurred in virtually all platelets and showed and identical, dotlike distribution. For immunoelectron microscopy we used protein A-colloidal gold on ultra-thin frozen sections to visualize the specific reaction indirectly. The staining for platelet factor 4, beta thromboglobulin, and fibrinogen localized exclusively over alpha-granules of washed platelets. Within the granules, platelet factor 4 was localized preferentially in the electron dense, alpha-granule nucleoid, whereas fibrinogen was more predominant in the electron-lucent granule periphery. Beta thromboglobulin localization did not show a preferential intragranular distribution.

Effect of heparin on in vivo platelet factor 4 (PF4) release and platelet aggregation after aspirin administration

Summary We studied the release in vivo of platelet specific proteins platelet factor 4 (PF4) and β-thromboglobulin (βTG), and confirmed that only PF4 is released, after heparin intravenous administration. A good correlation was found between platelet count and PF4 and this seems at variance with the idea that the source of released PF4 is vascular endothelium rather than platelets; however, such a possibility cannot be excluded. Although platelet function was blocked by aspirin injection heparin was still able to induce the release of PF4 and exerted a potentiating effect on platelet aggregation induced by ADP and collagen.