Platelet Heparin-Neutralizing Factor (Platelet Factor 4)

1975 ◽  
Vol 33 (01) ◽  
pp. 066-072 ◽  
Author(s):  
E. F Lüscher ◽  
R Käser-Glanzmann
Keyword(s):  
Molecular Weight ◽  
Basic Protein ◽  
Adenine Nucleotide ◽  
Human Platelets ◽  
Clotting System ◽  
Platelet Factor ◽  
High Molecular Weight Complex ◽  
Dense Bodies ◽  
Peptide Moiety ◽  
Platelet Aggregates

SummaryThe heparin-neutralizing platelet factor (PF4) is released from platelets under the influence of inducers of aggregation and nucleotide-release in the form of a high-molecular weight complex. The heparin-neutralizing activity is carried by a basic protein of molecular weight 29,700, four of which combine with a proteoglycan carrier, which in turn consists of 4 chondroitin sulfate A residues and a peptide moiety.The carrier-PF4-complex dissociates at higher ionic strength into proteoglycan and PF4 proper ; both forms (bound and free) are active in the inactivation of heparin, which displaces PF4 from its natural mucopolysaccharide carrier.PF4 is localized in human platelets in storage organelles, most likely in the α-granules and not in the adenine nucleotide and serotonin containing dense bodies. It is released simultaneously with the contents of these latter organelles.The physiological significance of PF4 is mainly seen in the interference with the inhibition of the clotting system by heparin within and in the vicinity of platelet aggregates. Released in circulation in the course of intravascular platelet damage, it may contribute to a facilitation of thrombin formation and action, by removing heparin.Adequately purified PF4 does not interact with fibrinogen and its derivatives and therefore is not directly involved in the induction of intravascular coagulation or platelet aggregation.

Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein

1981 ◽  
Author(s):  
A H Schmaier ◽  
J Kuchibhotla ◽  
R W Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Platelet Factor ◽  
Coagulant Activity

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.

Release Of Platelet Factor-4 In Vivo After Heparin Injection

1981 ◽  
Author(s):  
A Koneti Rao ◽  
John C Holt ◽  
Pranee James ◽  
Stefan Niewiarowski
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Plasma Samples ◽  
Platelet Factor ◽  
Immunoreactive Material ◽  
Elution Pattern

A single bolus of heparin administered to 8 normal volunteers resulted in a significant increase in levels in platelet poor plasma (PPP) of platelet factor-4 (PF4) but not low-affinity platelet factor-4/β-thromboglobulin (LA-PF4/βTG). However, the presence of heparin interfered with the binding of 125I-PF4 to antibody in radioimmunoassay (RIA). This effect was overcome by increasing the concentration of NaCl from 0.15 to 0.5 M in the buffer used for RIA. In order to establish that the increased amount of immunoreactive material present in PPP was indeed PF4, the protein was isolated from postheparin plasma. A bolus of 5000 units of porcine lung heparin (Upjohn) was administered intravenously to 2 volunteers and plasma samples obtained before and 5 minutes after the injection. The levels of PF4 in PPP rose from 18 and 10 ng/ml before to 185 and 454 ng/ml at 5 minutes after injection in the two volunteers, respectively. The 5 minute samples were adsorbed to heparin agarose columns and PF4 levels decreased to 16 and 10 ng/ml respectively. The immunoreactive material was eluted with 1.2 M NaCl from the heparin agarose columns, showing typical elution pattern for PF4. This material was applied to SDS-polyacrylamide gel electrophoresis in parallel with purified PF4 obtained from human platelets. RIA carried out on eluates from gel slices revealed a species of the same molecular weight as standard PF4. Thus, heparin injection results in appearance in the circulation of a material identical to PF4. LA-PF4/βTG and PF4 are located in same granules and released in parallel during platelet stimulation. Further, LA-PF4 is cleared from plasma 4 times slower than PF4. Therefore, the elevation of PF4alone suggests release from sites other than platelets.

scholarly journals Thrombocytin, A Novel Platelet Activating Enzyme from Bothrops Atrox (MARAJOENSIS) Venom

1977 ◽  
Author(s):  
S. Niewiarowski ◽  
E.P. Kirby ◽  
G.J. Stewart ◽  
R. Turna ◽  
M. Wiedeman ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Activate Factor ◽  
Factor X ◽  
Electron Microscopic ◽  
Platelet Factor ◽  
Small Vessels ◽  
Platelet Aggregates ◽  
Bothrops Atrox ◽  
Induced Aggregation

Thrombocytin (TCN) was purified from Bothrops atrox (BA) venom by precipitation with 1.2% Na-salicylate and chromatography on heparin-agarose column using increasing concentrations of lysine as eluent. It was homogeneous on SDS electrophoresis and had an apparent MW of 36,000. Immunoelectrophoresis with polyvalent anti-BA venom serum gave one cathodic arc indicating an isoelectric point higher than pH 8.6.TCN at a concentration of 1 yg/ml caused aggregation of human platelets, release of low affinity platelet factor 4 and serotonin, and stimulated platelets to retract fibrin.TCN was essentially free of fibrinogen clotting and fibrinolytic activities.TCN action on platelets was not mediated by the formation of thrombin since TCN did not activate Factor X or prothrombin and its action was not inhibited by hirudin.TCN is a serine protease since it was inhibited by DFP and it hydrolyzed a synthetic peptide, chromozyme UK (BZ-Val-Gly-Arg-pNA·HCl).TCN-induced aggregation of human platelets was completely inhibited by soy bean trypsin inhibitor, heparin, prostaglandin E1 and apyrase. Washed human platelets were 2-4 times less sensitive to TCN as compared to platelets in freshly prepared platelet rich plasma (PRP); their sensitivity to TCN gradually deteriorated during incubation of PRP at room temperature for 3 hours. Electron microscopic observations revealed formation of platelet aggregates characterized by pseudopod formation, centralization and partial loss of platelet granules. Infusion of TCN (3 yg) into the main artery of bat wing resulted in the formation of platelet aggregates seen on arterial and venous side which occasionally occluded small vessels.

Isolation Of Dense Bodies From Human Blood Platelets On Metrizamide Gradient

1981 ◽  
Author(s):  
F Rendu ◽  
M Lebret ◽  
J P Caen
Keyword(s):  
Plasma Membranes ◽  
Blood Platelets ◽  
Human Platelets ◽  
Release Mechanism ◽  
Inherited Disorders ◽  
Platelet Factor ◽  
Functional Studies ◽  
Dense Bodies ◽  
Serotonin Uptake And Release

In view of the prominent role of dense bodies in platelet activation suggested by the platelet dysfunctions observed in storage pool diseases, we have developed a method for the isolation of human platelet dense bodies, using mepa- crine to follow the purification.Each step of the purification (washing procedures, lysis and subcellular separation) has been controlled in order to obtain the minimum release of these granules. Platelet lysates were centrifuged on a short two step discontinuous metrizamide gradient which allowed the attainment of a high density pellet. This pellet consisted of isolated mepacrine fluorescent granules which showed the typical appearance of dense bodies by electron microscopy. The granule pellet was relatively free from plasma membranes as estimated by the remaining (3H) -concanavalin A or 125I after labelling the whole platelets before the fractionation. The low contamination by other granule populations was estimated by the different assayed markers, β-glucuronidase, monoamine oxidase and platelet factor 4. The method is simple, reproducible and allows the highest enrichment in dense bodies obtained until now with human platelets(x 170 enrichment in calcium and x 110 enrichment in (14C) 5-HT after labelling the whole platelets as compared to the homogenate). Functional studies performed with the isolated granules showed a rapid accumulation of (14C)-5-HT, and the initial uptake was inhibited by reserpine but remained insensitive to imipramine.The technique can be applied to the study of inherited disorders where the serotonin uptake and release mechanism has to be clarified.

Human Platelet Storage Organelles A Review

1977 ◽  
Vol 38 (04) ◽  
pp. 0963-0970 ◽  
Author(s):  
Miriam H. Fukami ◽  
Leon Salganicoff
Keyword(s):  
Secretory Granules ◽  
Subcellular Fractionation ◽  
Calcium Pyrophosphate ◽  
Human Platelets ◽  
Acid Hydrolases ◽  
Vascular Permeability Factor ◽  
Extracellular Medium ◽  
Platelet Factor ◽  
Platelet Storage ◽  
Dense Bodies

SummaryPlatelets contain numerous electron-dense subcellular organelles which have been referred to in the literature by various names such as α-granules, electron-dense and very electrondense granules, lysosomes, dense bodies, etc. Most of the organelles are secretory granules, since induction of secretion by appropriate stimuli causes degranulation of platelets and the appearance of the granule contents in the extracellular medium. Among the substances that are known to be stored and secreted by platelets are: serotonin, ATP, ADP, calcium, pyrophosphate, acid hydrolases, fibrinogen, vascular permeability factor, β-thromboglobulin, platelet factor 4 and growth factor.The recent literature concerning the localization of the secreted substances within specific platelet organelles is reviewed here. Results from electron microscopy and microprobe analysis, selective secretion experiments, subcellular fractionation studies and studies on platelets from patients with storage pool deficiency indicate that there are as many as four types of storage organelles in human platelets.

ADP And Epinephrine-Induced Release Of Platelet Fibrinogen

1981 ◽  
Author(s):  
K L Kaplan
Keyword(s):  
Adenine Nucleotide ◽  
Human Platelets ◽  
Dense Granule ◽  
Protein Release ◽  
Fibrinogen Concentration ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Added fibrinogen is said to be essential for induction of platelet aggregation and release by ADP and epinephrine. Furthermore, both ADP and epinephrine induce binding of fibrinogen to the platelet surface. In the present study gel-filtered human platelets were examined to determine whether they would aggregate and release platelet fibrinogen in response to ADP or epinephrine without exogenous fibrinogen. Platelets gel-filtered into Tyrode’s buffer containing ImM Mg++, no added Ca++, and 0.35% bovine serum albumin had a fibrinogen concentration in the supernatant of less than 1 nM. After aggregation by ADP or epinephrine the fibrinogen concentration in the supernatant ranged from 11 to 41 nM. ADP and epinephrine each induced biphasic aggregation and release of platelet factor 4 and β-thromboglobulin as well as of fibrinogen. Protein release by epinephrine was coincidental with dense granule adenine nucleotide release. Because Ca++ ions affect fibrinogen binding to platelets, the effect of Ca++ on aggregation and protein release was examined and it was found that both were inhibited by added Ca++ at 1-2 mM. The ability of gel-filtered platelets to undergo ADP and epinephrine induced aggregation and release in the absence of exogenous fibrinogen suggests that released platelet fibrinogen can support these processes. The concentrations of released fibrinogen in these experiments were lower than those reported to be necessary for exogenous fibrinogen for support of aggregation, suggesting that released fibrinogen may interact more efficiently with the platelet membrane. The amount of released fibrinogen in these experiments is similar to the Kd for high-affinity fibrinogen binding reported by Niewiarowski et al. (30nM). Finally, although inhibition of ADP and epinephrine induced aggregation and release by physiologic Ca++ concentrations implies that these processes do not occur in vivo, it is likely that platelet fibrinogen released by collagen or thrombin does function physiologically.

Aggregation of Fibrinogen and Its Degradation Products by Basic Proteins

1971 ◽  
Vol 25 (03) ◽  
pp. 566-579 ◽  
Author(s):  
G. J Stewart ◽  
S Niewiarowski
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
High Specificity ◽  
Platelet Factor 4 ◽  
Cationic Protein ◽  
Platelet Factor ◽  
Basic Proteins ◽  
Fibrin Degradation Products ◽  
Platelet Aggregates ◽  
Clinical Conditions

SummaryFibrinogen and early fibrinogen degradation products (FDP) of plasmin digestion containing Fragment XfP gave highly ordered polymers of limited branching with protamines and synthetic polylysine (molecular weight 175,000). Histones, polylysine of molecular weight 2,500, granulocyte cationic protein and platelet factor 4 formed amorphous precipitates with fibrinogen and early FDP. Fibrin degradation products (fdp) containing complexes of Fragment X° formed highly ordered polymers of extensive branching with these basic proteins.The splitting off of fibrinopeptides from fibrinogen and Fragment XfP was not essential for the formation of highly ordered polymers. However, in the presence of fibrinopeptides, high specificity of polymerizing agents such as basic protein is required. In the absence of fibrinopeptides, fibrin monomer or Fragment X° could polymerize after dissociation from complexes with other degradation products by any of the basic proteins.It has been suggested that the polymerization of fibrinogen and its derivatives by basic proteins may be an important factor contributing to the formation and deposition of fibrin-like material in clinical conditions connected with the destruction of tissues (release of histones), damage of granulocytes (release of lysosomal cationic protein) and intravascular formation of platelet aggregates (release of platelet factor 4).

scholarly journals Effects of the neutrophil-activating peptide NAP-2, platelet basic protein, connective tissue-activating peptide III and platelet factor 4 on human neutrophils.

1989 ◽  
Vol 170 (5) ◽  
pp. 1745-1750 ◽  
Author(s):  
A Walz ◽  
B Dewald ◽  
V von Tscharner ◽  
M Baggiolini
Keyword(s):  
Connective Tissue ◽  
Basic Protein ◽  
Human Platelet ◽  
Reversed Phase ◽  
Platelet Factor 4 ◽  
Human Neutrophils ◽  
Neutrophil Chemotaxis ◽  
Platelet Factor ◽  
Activated Platelets ◽  
Platelet Aggregates

Platelet basic protein (PBP), connective tissue-activating peptide III (CTAP-III), and platelet factor 4 (PF-4) were purified from human platelet release supernatants by heparin-Sepharose ion-exchange and reversed-phase HPLC, and their neutrophil-activating effects were compared with those of NAP-2, a peptide of 70 amino acids corresponding to part of the sequence of PBP (1) and with sequence homology to NAF/NAP-1. NAP-2-induced elastase release and a rise in cytosolic free Ca2+ at concentrations between 0.3 and 100 nM, and neutrophil chemotaxis at concentrations between 0.03 and 10 nM. It was half as potent as NAF/NAP-1 in inducing exocytosis but showed the same activity in the other responses. By contrast, only minimal if any effects were obtained with PBP, CTAP-III, and PF-4 up to 100 nM. NAP-2 thus appears to behave like a typical chemotactic receptor agonist. It could be generated from PBP and/or CTAP-III released from activated platelets and lead to the accumulation of neutrophils in platelet aggregates.

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