Megakaryocyte/Platelet-Restricted FVIII Expression Using a Platelet Factor 4 Promoter: Determination of Activation State and In Vivo Efficacy during Thrombocytosis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1142-1142
Author(s):  
Andrea L. Damon ◽  
Jolyon Jesty ◽  
Lesley E. Scudder ◽  
Dmitri V. Gnatenko ◽  
Wadie F. Bahou
Keyword(s):  
Hemophilia A ◽  
Storage Proteins ◽  
Coagulation Factor ◽  
Platelet Factor 4 ◽  
P Value ◽  
Factor V ◽  
Assay Sensitivity ◽  
Platelet Factor ◽  
Altered Expression

Abstract Ectopic delivery of coagulation factor VIII (FVIII) to megakaryocytes (Mk) represents a viable approach for localized tenase generation by effectively concentrating the FVIIIa/FIXa enzyme-cofactor complex onto the negatively-charged phospholipid surface of activated platelets. While phenotypic correction has been demonstrated using hemophilia A (FVIII−/−) murine models in vivo, the activation state of platelet FVIII (pFVIII), optimal promoter choice, and phenotypic correction in the setting of a thrombocytotic stimulus remain unestablished. Preliminary microarray experiments using human platelets (N=5) demonstrated that the Mk-specific platelet factor 4 (PF4) transcripts were among the most abundant, prompting use of the 1.1 Kb PF4 promoter for Mk/platelet-restricted expression of human B-domain-deleted (hBDD) FVIII within the background of an exon 17-deleted mouse model of hemophilia A (PF4/hBDD/FVIII−/−). A chromogenic tenase assay using gel-filtered platelets from PF4/hBDD/FVIII−/− mice confirmed the presence of functional FVIII equivalent to 73 mU·1x109 platelets·mL−1 (N = 10 mice). In contrast, FVIII was not detectable in PF4/hBDD/FVIII−/− plasma (assay sensitivity <20 pM) or in platelets from FVIII−/− mice. The ectopic pFVIII did not affect the release and/or function of other a-granule storage proteins as established by parallel measurements of platelet factor V (FV) using a prothrombinase assay. Paired tenase assays (± thrombin) confirmed that pFVIII (unlike pFV) required thrombin cleavage for complete activation. To delineate the effects of a thrombocytotic stimulus on pFVIII expression and/or function, PF4/hBDD/FVIII−/− (N = 10) or FVIII−/− control mice (N = 5) were injected with thrombopoietin (TPO; 10μg/kg/day for 5 days) resulting in an 87% average increase in platelet count. Day 10 tenase assay of TPO-injected PF4/hBDD/FVIII−/− mice demonstrated a 66% reduction in pFVIII activity (25 mU FVIII·1x109 platelets·mL−1), unassociated with altered expression of a-granule-stored amyloid-b-precursor protein (AbPP) as a control for storage granule content; plasmatic FVIII remained undetectable. In contrast, the decrease in total platelet FVIII biomass was less pronounced in TPO-treated PF4/hBDD/FVIII−/− mice, representing a 35% reduction from 147.6 mU to 96 mU after TPO stimulation. The decreased pFVIII in TPO-stimulated PF4/hBDD/FVIII−/− mice correlated with loss of phenotypic correction as evaluated using tail bleeding survival rates: wild-type mice (100%; N= 5), FVIII−/− (0%; N=5), PF4/hBDD/FVIII−/− (TPO-naïve 60%; N=11), PF4/hBDD/FVIII−/− (TPO-treated 0%; N=7) (p value between PF4/hBDD/FVIII−/− mice with and without TPO stimulation = 0.002). While these data establish that Mk-directed pFVIII (unlike pFV) is proteolytically inactive upon platelet activation, they also imply that thrombocytotic stimuli negatively affect the pFVIII bioavailability and phenotypic efficacy. As importantly, the hemostatic efficacy of platelet FVIII correlates best with localized platelet FVIII delivery (FVIII concentration/platelet) and not the systemic platelet FVIII bioavailability.

Thromboelastography in Children with Coagulation Factor Deficiencies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2139-2139 ◽  
Author(s):  
Meera B. Chitlur ◽  
Indira Warrier ◽  
Madhvi Rajpurkar ◽  
Wendy Hollon ◽  
Lolita Llanto ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Response To Treatment ◽  
Factor Vii ◽  
P Value ◽  
Factor V ◽  
Mean Values ◽  
Significant Difference

Abstract The thromboelastograph produces a continuous profile of the rheological changes that occur during the process of coagulation using whole blood. This information can be transformed into a dynamic velocity profile of the changes in blood elasticity occurring during clotting. We used the TEG® hemostasis analyzer in patients with hemophilia A or B with and without inhibitors and other coagulation factor deficiencies (OFD), to study the thromboelastographic profiles in these patients. Materials and Methods: 62 children (6 months-19 years old) were enrolled according to IRB regulations. 29 children had severe hemophilia A (SHA), 4 moderate hemophilia A or B (Mod.H), 2 severe factor VII deficiency, 1 combined factor V and VIII deficiency, 1 VWD (type II B), 1 severe factor V deficiency, 1 Severe PAI deficiency, 19 normal controls (NC), and 4 SHA with inhibitors (SHA+I). All patients were studied 72 hours after the last dose of factor. Citrated whole blood was activated using recombinant human tissue factor (Innovin, Dade Behring Inc®) and recalcified using 0.2M CaCl2. In patients with central lines with heparin, a heparinase cup was used. The TEG® was run for ≥ 90 min. CBC with differential was obtained on all subjects. Results: There was no significant difference in the CBC parameters among patients. Analysis of the TEG data revealed the following: Table 1 TEG Parameters (mean values) SHA (n=29) Mod.H (n=4) SHA+I (n=4) OFD (n=6) Control(n=19) MTG:Max rate of thrombin generation; TMG: Time to MTG; R: Reaction Time; K: Time to reach an amplitude of 20mm; MA: Max. Amplitude MTG(mm*100/sec) 8.7 9.6 1.3 9 17 TMG(min) 27.5 16.6 62.7 17.5 8.9 R(min) 22 14 56 15 7 K(min) 7 4 41 4 2 Max.Amplitude, MA (mm) 59 56 12 58 62 The rate of thrombin generation as visualized by plotting the 1st derivative of the TEG course, in patients with SHA without inhibitors, showed that they could be divided into 2 groups based on MTG (</>9). When analysed the 2 groups showed the following characteristics (5 representative curves from each group are shown): Figure Figure Table 2 TEG Parameters (Mean values) MTG < 9 (n=16) MTG > 9 (n=13) p value TMA: Time to MA; MTG(mm*100/sec) 5.5 12.6 <0.001 TMG (min) 33 20 0.009 R(min) 26 16 0.004 K(min) 9 3.4 0.03 MA(mm) 56.1 62.3 0.01 TMA(min) 60 38 0.006 13/29 children with SHA had target joints and 69%of patients with target joints had a MTG<9. Conclusions: SHA patients have variable bleeding tendencies as seen by the variation in MTG. A lower MTG is associated with a higher incidence of target joints. This may provide a clue as to which patients may have the greatest benefit from primary prophylaxis. Patients with OFD have a TEG® profile similar to Mod.H patients. SHA+I have poor thrombin generation as seen by a significantly longer TMG and R time (p <0.05), compared to all subjects. The TEG may provide valuable clues to the severity of bleeding tendencies in patients with factor deficiencies. In additional observations (not shown), it appears that the TEG may be used to monitor the response to treatment with factor concentrates and tailor treatment with rFVIIa.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Release of Platelet Factor 4 in Vivo during Intravascular Coagulation and in Thrombotic States

1968 ◽  
Vol 19 (03/04) ◽  
pp. 578-583 ◽  
Author(s):  
R Farbiszewski ◽  
S Niewiarowski ◽  
K Worowski ◽  
B Lipiński
Keyword(s):  
Coronary Thrombosis ◽  
Laboratory Diagnosis ◽  
Tolerance Test ◽  
Platelet Factor 4 ◽  
Thrombin Time ◽  
Significant Elevation ◽  
Platelet Factor ◽  
Thrombotic Diseases ◽  
Disseminated Intravascular Clotting

SummaryPlatelet factor 4 released from platelets into the circulating blood was determined using both the heparin thrombin time and paracoagulation methods. It has been found that thrombin injected intravenously into rabbits releases large amounts of this factor. Infusion of plasmin does not release this factor and this finding may be of importance for the differential diagnosis between disseminated intravascular clotting and primary fibrinolysis. PF4 is not released during the hyper coagulable condition induced by HgCl2 intoxication. Only small amounts of this factor are released after contact factor infusion.A significant elevation of extraplatelet PF4 was found in 23 patients with fresh coronary thrombosis and in 9 patients with thrombophlebitis and thromboembolic complications.The significance of the above findings for the pathogenesis, treatment and laboratory diagnosis of thrombotic diseases with particular reference to heparin tolerance test is discussed.

Clearance and In Vivo Release by Heparin of Human Platelet Factor 4 (PF4) in the Rabbit

1984 ◽  
Vol 52 (02) ◽  
pp. 157-159 ◽  
Author(s):  
M Prosdocimi ◽  
N Scattolo ◽  
A Zatta ◽  
F Fabris ◽  
F Stevanato ◽  
...  
Keyword(s):  
Half Life ◽  
Human Platelet ◽  
Slow Component ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
In Vivo Release ◽  
Partial Release ◽  
Different Doses ◽  
New Zealand Rabbits

Summary13 male New Zealand rabbits were injected with two different doses (25 μg/Kg and 100 μg/Kg) of human platelet factor 4 antigen (PF4). The disappearance of the protein was extremely fast with an half-life for the fast component of 1.07 ± 0.16 and 1.76 ± 0.11 min respectively. The half-life for the slow component, detectable only with the highest dosage, was 18.8 min.The administration of 2500 I.U. of heparin 30 min after PF4 administration induced a partial release of the injected protein and its clearance from plasma was slow, with half-life of 23.3 ± 5.9 min and 30.9 ± 2.19 min respectively.

Detection of Enhanced In Vivo Platelet α-Granule Release in Different Patient Groups - Comparison of β-Thromboglobulin, Platelet Factor 4 and Thrombospondin Assays

1984 ◽  
Vol 52 (02) ◽  
pp. 183-187 ◽  
Author(s):  
D A Lane ◽  
H Ireland ◽  
S Wolff ◽  
E Ranasinghe ◽  
J Dawes
Keyword(s):  
Platelet Factor 4 ◽  
Renal Elimination ◽  
Elevated Plasma ◽  
Platelet Factor ◽  
Release Reaction ◽  
Heparin Anticoagulation ◽  
Patient Groups ◽  
Granule Release ◽  
Sensitive Marker

SummaryDuring the platelet release reaction β-thromboglobulin (βTG), platelet factor 4 (PF4) and thrombospondin (TSP) are released from the platelet into plasma and assays of these proteins can be used to monitor in vivo platelet activation. We have assessed their relative merits as markers of the in vivo platelet α-granule release reaction in a number of patient groups which have previously been shown to have elevated plasma βTG and/or PF4 levels. It is concluded that in diseases or conditions not complicated by its reduced clearance, βTG is the most sensitive marker of in vivo platelet α-granule release. However, the TSP assay may be the least ambiguous when monitoring the platelet α-granule release reaction in patients with renal failure who are undergoing haemodialysis with heparin anticoagulation. Under these circumstances plasma βTG, but not PF4 or TSP, levels are elevated because of impaired renal catabolism, and the presence of a heparin-releasable reservoir of PF4 on the endothelium complicates the use of the PF4 assay. In liver failure none of these assays may accurately reflect platelet α-granule release because of impaired hepatic or renal elimination of the proteins.

In vivo effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP on haemopoiesis of mice treated with 5‐fluorouracil

1996 ◽  
Vol 94 (3) ◽  
pp. 443-448 ◽  
Author(s):  
Sallouha Aidoudi ◽  
Martine Guigon ◽  
Isabelle Lebeurier ◽  
Jacques P. Caen ◽  
Zhong Chao Han
Keyword(s):  
Platelet Factor 4 ◽  
Platelet Factor

scholarly journals Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2180-2190
Author(s):  
MD Rand ◽  
M Kalafatis ◽  
KG Mann
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Factor V ◽  
Platelet Factor ◽  
Factor Va ◽  
Plasma Factor

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

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.

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