Functional Analyses of Patient-derived IgG Monoclonal Anticardiolipin Antibodies Using In Vivo Thrombosis and In Vivo Microcirculation Models

SummaryAntiphospholipid antibodies (aPL) have been associated with thrombosis and pregnancy losses in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. It has been proposed that aPL may affect endothelial cell (EC) function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. It has been proposed that aPL may affect EC cell function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. This study proposes to test the hypotheses that some IgG anticardiolipins (IgG aCL) with thrombogenic properties in mice, exert their effects through activation of endothelium. We studied seven patient-derived monoclonal aCL for their thrombogenic properties in an in vivo pinch-induced thrombosis model, and their functional activities in activating EC by analyzing in vivo leukocyte adhesion to endothelium in microcirculation in venules in exposed murine cremaster muscle and in vitro adhesion molecule expression in cultured EC. The binding of the monoclonal aCL to EC was also tested. In addition to the previous identified thrombogenic IS2, four of the five new more IgG monoclonal aCL (from two patients) were found to be thrombogenic. Of these five thrombogenic aCL, three caused more in vivo leukocyte adhesion to EC in microcirculation, as compared to that induced by the H2 control human monoclonal IgG, and enhanced expression of adhesion molecules (particularly VCAM-1) on cultured EC. These data show that about 2/3 patientderived IgG monoclonal aCL are thrombogenic and suggest that some thrombogenic IgG aCL exert their effects through activating EC.

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A Locally Administered Novel Heparin Complex Attenuates Collagen-Induced Platelet Activation and Thrombosis In Vivo,

Blood ◽

10.1182/blood.v118.21.3361.3361 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3361-3361

Author(s):

Riitta Lassila ◽

Annukka Jouppila ◽

Ulla M Marzec ◽

Stephen R Hanson

Keyword(s):

Blood Flow ◽

Platelet Aggregation ◽

Thrombus Formation ◽

Ptfe Graft ◽

Thrombin Time ◽

Thrombosis Model ◽

Baboon Model ◽

Induced Aggregation

Abstract Abstract 3361 We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002). Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen. In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008). In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600 Disclosures: Lassila: Aplagon: Chief Scientific Advisor.

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Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters

Blood ◽

10.1182/blood.v85.3.712.bloodjournal853712 ◽

1995 ◽

Vol 85 (3) ◽

pp. 712-719 ◽

Cited By ~ 23

Author(s):

H Deckmyn ◽

JM Stassen ◽

I Vreys ◽

E Van Houtte ◽

RT Sawyer ◽

...

Keyword(s):

Platelet Adhesion ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Femoral Vein ◽

Video Recording ◽

Hirudo Medicinalis ◽

Thrombosis Model

Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody- conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose- dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.

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Tetrahydrocurcumin Downregulates MAPKs/cPLA2 Signaling and Attenuates Platelet Thromboxane A2 Generation, Granule Secretion, and Thrombus Growth

Thrombosis and Haemostasis ◽

10.1055/s-0041-1735192 ◽

2021 ◽

Author(s):

Weiqi Li ◽

Yongjie Ma ◽

Chunmei Zhang ◽

Binlin Chen ◽

Xiandan Zhang ◽

...

Keyword(s):

Thrombus Formation ◽

Biological Activities ◽

Thromboxane A2 ◽

Mapk Signaling ◽

Inhibitory Effects ◽

Platelet Factor ◽

Thrombosis Model ◽

Granule Secretion

AbstractPlatelet granule secretion plays a key role in atherothrombosis. Curcumin, a natural polyphenol compound derived from turmeric, exerts multiple biological activities. The current study sought to investigate the efficacy of tetrahydrocurcumin (THC, the major active metabolite of curcumin) on platelet granule secretion in vitro and thrombus formation in vivo. We found that THC significantly attenuated agonist-induced granule secretion in human gel-filtered platelets in vitro, including CD62P and CD63 expression and platelet factor 4, CCL5, and adenosine triphosphate release. These inhibitory effects of THC were partially mediated by the attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation, leading to a decrease in thromboxane A2 (TxA2) generation. Moreover, the MAPK (Erk1/2, JNK1/2, and p38 MAPK) signaling pathways were downregulated by THC treatment, resulting in reduced cPLA2 activation, TxA2 generation, and granule secretion. Additionally, THC and curcumin attenuated murine thrombus growth in a FeCl3-induced mesenteric arteriole thrombosis model in C57BL/6J mice without prolonging the tail bleeding time. THC exerted more potent inhibitory effects on thrombosis formation than curcumin. Through blocking cyclooxygenase-1 activity and thus inhibiting platelet TxA2 synthesis and granule secretion with aspirin, we found that THC did not further decrease the inhibitory effects of aspirin on thrombosis formation. Thus, through inhibiting MAPKs/cPLA2 signaling, and attenuating platelet TxA2 generation, granule secretion, and thrombus formation, THC may be a potent cardioprotective agent.

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Functional Analyses of Patient-derived IgG Monoclonal Anticardiolipin Antibodies Using In Vivo Thrombosis and In Vivo Microcirculation Models

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614033 ◽

2000 ◽

Vol 84 (09) ◽

pp. 388-395

Author(s):

Xiaowei Liu ◽

Ricardo Espinola ◽

Tsawei Olee ◽

Min Zhu ◽

Nigel Harris ◽

...

Keyword(s):

Endothelial Cells ◽

Antiphospholipid Syndrome ◽

Adhesion Molecule ◽

Antiphospholipid Antibodies ◽

Cell Function ◽

Leukocyte Adhesion ◽

Anticardiolipin Antibodies ◽

Intercellular Adhesion Molecule ◽

Enzyme Linked Immunosorbent Assay

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Role of murine integrin α2β1 in thrombus stabilization and embolization: Contribution of thromboxane A2

Thrombosis and Haemostasis ◽

10.1160/th07-02-0089 ◽

2007 ◽

Vol 98 (11) ◽

pp. 1072-1080 ◽

Cited By ~ 24

Author(s):

Miroslava Pozgajova ◽

Judith Cosemans ◽

Imke Munnix ◽

Beate Eckes ◽

Bernhard Nieswandt ◽

...

Keyword(s):

Thrombus Formation ◽

Thromboxane A2 ◽

Wild Type ◽

Thrombosis Model ◽

Thromboxane Receptors ◽

Thromboxane A2 Analogue ◽

Α2β1 Integrin

SummaryPlatelets stably interact with collagen via glycoprotein (GP)VI and α2β1 integrin. With α2-null mice, we investigated the role of α2β1 in thrombus formation and stability in vivo and in vitro. Using a FeCl3-induced thrombosis model, in arteries from α2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A2 analogue U46619 was borderline effective in suppressing the embolisation in α2-null mice. In vitro, perfusion of α2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from α2-null mice. We conclude that integrin α2β1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A2 release. The increased embolization in α2-null mice may argue against the use of α2β1 integrin inhibitors for antithrombotic therapy.

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Thrombocytopathy leading to impaired in vivo haemostasis and thrombosis in platelet type von Willebrand disease

Thrombosis and Haemostasis ◽

10.1160/th16-04-0317 ◽

2017 ◽

Vol 117 (03) ◽

pp. 543-555 ◽

Cited By ~ 5

Author(s):

Harmanpreet Kaur ◽

Kathryn Corscadden ◽

Jerry Ware ◽

Maha Othman

Keyword(s):

Thrombus Formation ◽

Annexin V ◽

Blood Platelet ◽

Von Willebrand Disease ◽

Clot Formation ◽

Delayed Response ◽

Thrombosis Model ◽

Laser Injury

SummaryPlatelet defects due to hyper-responsive GPIbα causing enhanced VWF interaction, counter-intuitively result in bleeding rather than thrombosis. The historical explanation of platelet/VWF clearance fails to explain mechanisms of impaired haemostasis particularly in light of reported poor platelet binding to fibrinogen. This study aimed to evaluate the defects of platelets with hyper-responsive GPIbα and their contribution to impaired in vivo thrombosis. Using the PT-VWD mouse model, platelets from the hTgG233V were compared to control hTgWT mice. Platelets’ pro-coagulant capacity was evaluated using flowcytometry assessment of P-selectin and annexin V. Whole blood platelet aggregation in response to ADP, collagen and thrombin was tested. Clot kinetics using laser injury thrombosis model and the effect of GPIbα inhibition in vivo using 6B4; a monoclonal antibody, were evaluated. Thrombin-induced platelet P-selectin and PS exposure were significantly reduced in hTgG233V compared to hTgWT and not signifi-cantly different when compared to unstimulated platelets. The hTgG233V platelets aggregated normally in response to collagen, and had a delayed response to ADP and thrombin, when compared to hTgWT platelets. Laser injury showed significant impairment of in vivo thrombus formation in hTgG233V compared to hTgWT mice. There was a significant lag in in vitro clot formation in turbidity assay but no impairment in thrombin generation was observed using thromboelastography. The in vivo inhibition of GPIbα facilitated new – unstable – clot formation but did not improve the lag. We conclude platelets with hyper-responsive GPIbα have complex intrinsic defects beyond the previously described mechanisms. Abnormal signalling through GPIbα and potential therapy using inhibitors require further investigations.Supplementary Material to this article is available online at www.thrombosis-online.com.

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ANTITHROMBOTIC OLIGOAMINES

10.1055/s-0038-1643439 ◽

1987 ◽

Author(s):

K Rehse ◽

U Lukens ◽

S Leibring ◽

V Schein ◽

A Kesselhut

Keyword(s):

Thrombus Formation ◽

Coagulation Factors ◽

Thrombin Time ◽

Metabolic Pattern ◽

New Class ◽

Thrombosis Model ◽

A 23187 ◽

Shot Number

We have found that oligoamines of the general formula R2−3X(R=-(CH2)m-NH-(CH2)n-Y) in which X and Y may be aliphatic, alicyc-lic, aromatic or even heterocyclic moieties are a new class of compounds which exhibit platelet aggregation inhibiting and anticoagulant activities in vitro and have antithrombotic properties in vivo. The compound RE 1492 (N,N’,N1’-Tris-4-phenylbutylbenzene-1,3,5-trimethanamine) is chosen as example to demonstrate these effects. In PRP the following IC50 of RE 1492 (inductor in brackets) were measured: 3 ¼mol/L (Collagen), 1 ¼mol/L (ADP, 2ndphase), 7.5 μmol/L (ADP, lstphase), 2,5 μmol/L (A 23187, 2ndphase), 7,5 μmol/L (Ionophor A 23187, lstphase), 30 ¼mol/L (Thrombin). The inhibition of the aggregation Induced by ADP could as well be demonstrated in whole blood. The formation of fibrin was inhibited as shown by the prolongation of the thromboplastin time (Quick) and the partial thromboplastin time (PTT) the first being more sensitive (25% of normal at 50 ymol/L) than the latter (25% of normal at 100 ymol/L). The reason was the inhibition of coagulation factors in the following order: VII (25% of normal at 12.5 ¼mol/L) >WErwnr/IX (25 ¼mol/L) »X (200 μmol/L). The thrombin time remains normal. The antithrombotic properties of RE 1492 were investigated in an in vivo thrombosis model. The formation of platelet thrombi in mesenteric arterioles and venoles of rats (diameterM5 ym) was induced by a laser beam. In controls 1,76±1,14 (SD) shots (50 msec, 50 mW) on the arterioles were necessary for thrombus formation. Twenty minutes after i.v. application of RE 1492 this number rose to 3,18±2,08 (3 mg/kg, p ≤ 0,01, X2-test) and 4,59±1,93 (10 mg/kg, p ≤ 0,01) in arterioles. In venoles of the control animals 1,29±0,45 shots were necessary for thrombus formation. This number rose to 2,11±1,62 (p ≤ 0,05) after 3 mg/kg and 3,28±2,03 (p0,01) after 10 mg/kg. As the number of shots applied was limited to five an average shot number of 5± SD would indicate that no thrombus formation takes place at all. As RE 1492 does neither influence the metabolic pattern of arachidonic acid in platelets nor the activity of phosphodiesterase or adenylatcyclase it is supposed that the oligoamines exert their effects by interaction with phospholipids (PL) resulting in a “membrane stabilization” in platelets and inhibition of PL dependent coagulation factors during fibrin formation.

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Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645198 ◽

1990 ◽

Vol 63 (02) ◽

pp. 220-223 ◽

Cited By ~ 19

Author(s):

J Hauptmann ◽

B Kaiser ◽

G Nowak ◽

J Stürzebecher ◽

F Markwardt

Keyword(s):

Factor Xa ◽

Thrombin Inhibitors ◽

Factor Xa Inhibitors ◽

Venous Stasis ◽

Clotting Time ◽

Thrombosis Model ◽

Activity Factor ◽

Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

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A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650089 ◽

1980 ◽

Vol 44 (02) ◽

pp. 081-086 ◽

Cited By ~ 8

Author(s):

C V Prowse ◽

A E Williams

Keyword(s):

Platelet Rich Plasma ◽

Thrombus Formation ◽

Factor Ix ◽

Coagulation System ◽

In Vitro Tests ◽

Clinical Use ◽

Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661675 ◽

1986 ◽

Vol 56 (03) ◽

pp. 318-322 ◽

Cited By ~ 18

Author(s):

V Diness ◽

P B Østergaard

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Thrombus Formation ◽

Experimental Models ◽

Protamine Sulfate ◽

Low Molecular Weight ◽

Antithrombotic Effect ◽

Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

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