Interaction between Histidine-Rich Glycoprotein and Platelet Factor 4 with Dermatan Sulfate and Low-Molecular-Weight Dermatan Sulfate

Abstract Context.—It is now widely accepted that the pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated by the generation of a wide array of functional and molecularly heterogeneous anti–heparin-platelet factor 4 (AHPF4) antibodies that may mediate platelet and/or endothelial cell activation/destruction. Objective.—We investigated the differential prevalence and functionality of AHPF4 immunoglobulin subtypes (IgA, IgG, and IgM) in plasmas obtained from orthopedic patients immobilized with Plaster-Cast and treated with clivarin (a low-molecular-weight heparin) in comparison to a placebo for the prophylaxis of deep-vein thrombosis. Design and Methods.—Clivarin was administered subcutaneously at a fixed daily dosage of 1750 U without any adjustment or loading dosage. Citrated plasmas were obtained at baseline, at 10 to 14 days, and at postbrace procedure (5–12 weeks). An enzyme-linked immunosorbent assay (ELISA) was used to quantitate the AHPF4 antibody titers. The functionality of the ELISA-positive samples was determined by a 14C-serotonin release assay (SRA). Results.—In the ELISA test, 16 of 1073 samples (1.5%; 6 in clivarin and 10 in placebo groups) were positive for AHPF4 antibodies (mean optical density [OD] = 0.46 ± 0.02). None of the ELISA-positive samples for AHPF4 antibodies could mediate platelet activation responses as determined by the SRA (0%–3% serotonin release, P > .10, n = 16). Through differential immunoglobulin subtype analysis of the samples positive for (cumulative) AHPF4 antibodies, we determined that their relative prevalence in plasma were as follows: IgM (mean OD = 0.71 ± 0.13) > IgG (0.31 ± 0.08) > IgA (0.14 ± 0.02). Although there was no significant difference in the total antibody titers between clivarin and placebo groups, the antibody subtyping data showed conversion trends (ie, IgA [clivarin to placebo], IgG [placebo to clivarin], and IgM [clivarin to placebo]). Conclusion.—These observations indicate that even at reduced dosages, clivarin can shift the immunogenic up-regulation toward the IgG subpopulation; however, the IgG subtype is of a nonfunctional type of AHPF4 antibody and thus may not cause any HIT-related pathogenic responses.

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NEUTRALIZING EFFECT OF PLATELET FACTOR 4 OR HISTIGINE-RICH GLYCOPROTEIN AGAINST LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN

10.1055/s-0038-1643499 ◽

1987 ◽

Author(s):

K Takahashi ◽

M Niwa ◽

N Sakuragawa

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Antithrombin Iii ◽

Platelet Factor 4 ◽

Low Molecular Weight ◽

Bleeding Tendency ◽

Human Origin ◽

Platelet Factor ◽

At Iii ◽

Antifactor Xa

Purpose: Low molecular weight(LMW) heparin shows stronger antifactor Xa(F-Xa) and weaker anti-thrombin(TH) activities compared with unfractionated(UF) heparin, and shows less bleeding tendency in the cases of clinical use. Platelet factor 4(Pf-4) and histidine-rich glycoprotein(HRG) neutralize heparin. We investigated on the heparin neutralizing effects of them to both kinds of heparinMaterials and methods: LMW heparin(Kabi and Pharmuka) and UF heparin(Novo) were used. Antithrombin III(AT-III), HRG(human origin ) and pf-4( bovine origin ) were purified by our methodsTH(Green-Cross) and F-Xa(Sigma) were used. Reaction mixtures for anti-TH or anti-F-Xa were as follows: 1 vol of AT-III( 0.1 U/ml)+ 1 vol of heparin( 10 ug/ml)+l vol of pf-4 or HRG(varied)→incubated for 5 min→+l vol of TH(5 U/ml) or F-Xa( 7 nKat/ml)→incubated for 5 min→ + S-2238 or S-2222→ recorded at 405 nm.Results: (1) Pf-4 showed the equivalent anti-TH effect on both kinds of heparin, and 3 ug of pf-4 neutralized 1 ug of heparinOn F-Xa neutralizing effect, 13 ug of pf-4 neutralized 1 ug of UF heparin, but could not neutralize LMW heparin. (2) HRG showed the same results on anti-TH effect of both kinds of heparin, but could not neutralize the anti-F-Xa effect of LMW heparin on the same amount of HRG which neutralized that of UF heparin. Conclusion: Anti-F-Xa effect of. LMW heparin could not be easily neutralized by pf-4 or HRG compared with that of UF heparin.

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Mechanism of low-molecular-weight heparin reversal by platelet factor 4

Thrombosis Research ◽

10.1016/j.thromres.2008.12.047 ◽

2009 ◽

Vol 124 (1) ◽

pp. 149-155 ◽

Cited By ~ 6

Author(s):

Martine M. Fiore ◽

Ian M. Mackie

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Platelet Factor 4 ◽

Low Molecular Weight ◽

Platelet Factor

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Molecular and Functional Heterogeneity in Contaminants Isolated from Recalled Heparin. Impact on Anticoagulation and Potential Adverse Reactions

Blood ◽

10.1182/blood.v112.11.4048.4048 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4048-4048

Author(s):

Jawed Fareed ◽

Debra Hoppensteadt ◽

Walter Jeske ◽

Cafer Adiguzel ◽

Omer Iqbal ◽

...

Keyword(s):

Molecular Weight ◽

Whole Blood ◽

Thrombin Generation ◽

Adverse Reactions ◽

Dermatan Sulfate ◽

Platelet Factor 4 ◽

Contact Activation ◽

Clotting Time ◽

Platelet Factor ◽

Chromatographic Methods

Abstract The main contaminant in the recalled batches of unfractionated heparin (UFH) is reported to be oversulfated chondroitin sulfate (OSCS). No data on the composition and biologic properties of this reported OSCS is available. Moreover, it has been assumed that different batches of preparations contained similar forms of OSCS. Since this contaminant has been purposefully added to UFH and has been found in several batches from various suppliers and is also found in some of the low molecular weight heparins (LMWHs), it was hypothesized that some type of OSCS was used to supplement UFH preparations. To test this hypothesis four batches of contaminated UFH and two batches of LMWH were investigated. Four batches of UFH of the recalled products from the US suppliers and two batches of a LMWH from the European community were compared. The molecular weight profile studies were carried out using high performance liquid chromatographic methods. Each of these products were subjected to heparinase-1 digestion to determine the non-digestable components. The anticoagulant activities of each of these products were measured using the whole blood activated clotting time and thromboelastographic analysis. The effect of each product was also studied on thrombin generation (Fibrinopeptide A, thrombin antithrombin complex, and prothrombin F1.2) and contact activation. The anticoagulant assays (PT, APTT, TT, Heptest, ecarin clotting time), antiprotease profile (anti-Xa and anti-IIa) and thrombin generation inhibitory studies were carried out in the normal human plasma. Protamine and platelet factor 4 neutralization were also carried out in plasma. Non-heparin contaminants were also isolated from each of the heparins by digestion of heparin followed by alcohol precipitation and ion exchange chromatographic methods. The isolated components were further purified by physicochemical methods. Each of these components were profiled for the molecular, structural and biologic activity. In addition each of the contaminants were further characterized in terms of their interactions with SERPINS (AT III and HCII), platelet factor 4 and analyzed for anticoagulant activity in whole blood and citrated plasma systems. The four contaminated UFHs (H1-H4) did not exhibit any major differences in the molecular weight profile (14.8–15.6 KDa). The USP potency of these products were also similar (158–170 U/mg). The anticoagulant actions were comparable in the different whole blood and global assays. However, the anti-Xa and anti-IIa ratios were found to exhibit some variations (0.93–1.24). Each product showed differences in the heparinase resistant component (14–30%). H1 and H3 products also contained significant amounts of dermatan sulfate. Each of the contaminants exhibited different neutralization profiles with PF4 and protamine sulfate. The LMWH products were comparable in all of the studies including the molecular weight profile and biologic actions, however, they showed differences in the heparinase-1 digestion profiles. Moreover, the molecular weight of the contaminant obtained from the LMWH was lower (12.8 vs. 14.1–16.8 KDa). The contaminants also exhibited differences in thrombin generation markers. The USP potency of the heparin contaminants varied from 28–46 U/mg whereas the contaminant from the LMWH exhibited a potency of 38–46 USP U/mg. These studies suggest that the contaminants isolated from recalled batches of heparin are heterogenous in nature and may originate from multiple sources. Moreover, the contaminants obtained form LMWHs may exhibit additional structural and biologic differences. Therefore, the wide variations observed in the adverse reactions with recalled heparins may be due to compositional variations in the contaminants.

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A prospective analysis of heparin–platelet factor 4 antibodies in pregnant women treated with the low-molecular-weight heparin, dalteparin

Blood Coagulation & Fibrinolysis ◽

10.1097/mbc.0b013e3282f102ea ◽

2008 ◽

Vol 19 (6) ◽

pp. 477-481 ◽

Cited By ~ 1

Author(s):

Frank Gerdsen ◽

Beate Luxembourg ◽

Florian Langer ◽

Rupert Bauersachs ◽

Edelgard Lindhoff-Last

Keyword(s):

Molecular Weight ◽

Pregnant Women ◽

Low Molecular Weight Heparin ◽

Platelet Factor 4 ◽

Low Molecular Weight ◽

Prospective Analysis ◽

Platelet Factor

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Effect of Histidine-rich Glycoprotein and Platelet Factor 4 on the Interaction Between Low Molecular Weight Heparin and Antithrombin III

Japanese Journal of Thrombosis and Hemostasis ◽

10.2491/jjsth.4.423 ◽

1993 ◽

Vol 4 (6) ◽

pp. 423-429

Author(s):

Takayuki SUGIYAMA ◽

Mari ITOH

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Antithrombin Iii ◽

Platelet Factor 4 ◽

Low Molecular Weight ◽

Platelet Factor

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Heparin-Induced Thrombocytopenia: Prevalence in a Large Cohort of Patients and Confirmed Role of PF4/Heparin Complex as the Main Antigen for Antibodies

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/107602969700300309 ◽

1997 ◽

Vol 3 (3) ◽

pp. 203-209 ◽

Cited By ~ 3

Author(s):

Fabrizio Fabris ◽

Immacolata Cordiano ◽

Federica Salvan ◽

Leopoldo Saggin ◽

Giuseppe Cella ◽

...

Keyword(s):

Molecular Weight ◽

Platelet Count ◽

Platelet Factor 4 ◽

Enzyme Linked Immunosorbent Assay ◽

Low Molecular Weight ◽

Dependent Manner ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Heparin Treatment ◽

Daily Dose

We performed a retrospective study on the prevalence of heparin-induced thrombocytopenia (HIT) in 233 patients receiving hog mucosa heparin therapy. Of these, 82 patients received s.c. calcium heparin, 130 patient received unfractionated (UF) i.v. heparin, and 21 patients received low molecular weight heparins (LMWH). An additional four patients, referred to our consultation and diagnosed by us as having clinically active type II HIT (HIT-II) were also studied. The mean platelet count of the 233 patients receiving heparin showed a significant decrease after 2 days of heparin treatment and a following significant increase 6 days later (basal: 257 ± 147 x 109 platelets/L; day 2: 239 ± 122, p < 0.0002; day 6: 286 ± 119, p < 0.004). Of the 212 patients receiving UF heparin, 13 (6%) fulfilled the criteria for HIT-II: seven of these had received i.v. heparin (mean daily dose 26,600 ± 4,082 IU ± SD) and six had received s.c. heparin (mean daily dose 21,428:t 6,900 IU). Their mean basal platelet count was 226 ± 100 SD × 109 platelets/L and the nadir during heparin treatment was 78 ± 39 x 10 9 platelets/L. Thrombotic complications occurred in four (30.7%) of the 13 patients with HIT-II. Since the immunological mechanism has been demonstrated for HIT-II and since platelet factor 4 (PF4) was identified as the co-factor for the binding of heparin-related antibodies, we set up our own enzyme-linked immunosorbent assay (ELISA) for testing antibodies against PF4/heparin complex bound through electrostatic bridges to the solid phase. The highest binding capacity of HIT-related IgG to the multimolecular complex was obtained at 20 μg/ml for PF4 and 3 μg/ml for heparin, corresponding to 250 ng of PF4 and 42 ng of heparin in each microtiter well. Such binding was inhibited in a dose-dependent manner by increasing amounts of heparin, protamine hydrochloride, and a monoclonal antibody anti-human PF4 clone 1OB2. We observed that HIT-related antibodies bound also to PF4/LMWH complexes but the optimal PF4/glycosaminoglycan ratio appeared more critical for LMWH (enoxaparin, fraxiparin, and pamaparin) than for UF heparin. Sera from eight patients with HIT-II were tested by PF4/heparin ELISA; six of these had IgG against the complex PF4/heparin and three also had IgM. The persistence of HIT-related antibodies was investigated in three patients: in one such antibodies were still detectable 3 years after the acute episode, while in the other two, they disappeared after 6 months and 1 year, respectively. Key Words: Heparin-related anti body—Platelet factor 4 (PF4)—Heparin—Low molecular weight heparin—Thrombocytopenia—Thrombosis.

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