Catabolism of low-dose heparin in man

An iodinated heparin derivative was injected with carrier heparin into healthy volunteers. The iodinated material was of similar molecular weight to the underivatised heparin, highly sulphated, and biologically active. It bound to plasma proteins including antithrombin III $\text{in vivo}$, and more than 85% was firmly bound to platelet factor 4 or protamine $\text{in vitro}$. After injection of 1–100 units, 80–90% was removed from the plasma within 40 minutes, most of it being sequestered by the liver and some by the spleen, lungs, and kidneys. Iodinated material was then again released into the plasma. The molecular weight of this product was unchanged, but it was no longer biologically active. It was not bound to antithrombin III $\text{in vivo}$, or to platelet factor 4 and protamine $\text{in vitro}$, and it was markedly desulphated. Except at doses greater than 1,000 units, the labelled material was degraded to small fragments before excretion in the urine.     

A train difference in anticoagulant response to subcutaneous administration of heparin in mice

A significant difference in anticoagulant response to subcutaneous administration of heparin between two strains of mice has been discovered. Agouti C3H mice had a faster, greater and more prolonged response than albino Swiss mice. On addition of heparin to the blood of the two strains $\text{in vitro}$, identical clotting times were obtained. There was no significant difference in anticoagulant response to intravenous injection in the two strains of mice for a quantity of heparin which gave blood levels similar to those obtained with subcutaneous injection in the Swiss mice. The difference in response to subcutaneous heparin in these two strains of mice can be obtained at different injection sites. The C3H mice also showed greater anticoagulant response than the Swiss mice, after introducing heparin together with citric acid into the small intestine. Injection of ³⁵S-heparin in the foot pad subcutaneously demonstrated that the removal of radioactivity from the injection site was faster and the plasma level of radioactivity was higher in C3H mice than Swiss mice. These results indicate a strain difference in the transfer of heparin to the circulation after subcutaneous or intestinal administration and suggest this provides a suitable animal model for study of absorption mechanisms for heparin.     

Variation in commercial heparin and its relation to the problem of heparin standardization for clinical use

This paper reports values for chemical and biological analyses for eight recently obtained commercial heparins, an acetic acid treated heparin and sheep heparin and compares these with values found in a previous study. While there is less variation in chemical analytical values, a lower sulfur content and higher acetyl value is characteristic. When potency estimations were determined by $\text{in vitro}$ assay systems for heparin, inconsistency was found in potency values obtained. The results obtained were compared with values obtained with an $\text{in vivo}$ assay in dogs, similar to clinical control methods for heparin and showing on statistical testing adequate design and reliability. A beef lung heparin and a pig mucosa heparin preparation showed the same potency by these $\text{in vivo}$ tests, while a sheep lung heparin and pig mucosa heparin partially inactivated by exposure to acetic acid show much lower potencies. USP, BP, Howell assays did not give these results for this series of heparin preparations and thus do not predict their potency $\text{in vivo}$. Two types of pmr spectra of heparin are observed, those with an N-acetyl peak such as pig mucosa heparin, and those with no N-acetyl peak, such as beef lung heparin. Beef lung heparin appears to have one component on microelectrophoresis while pig mucosa heparin in most cases has two. The importance of these results for the standardization of heparin are discussed.     

Purification and biological property of heparin cofactor II: Activation of heparin cofactor II and antithrombin III by dextran sulfate and various glycosaminoglycans

Heparin cofactor II (HC II) has been purified from human plasma by a mocification of the method described by Tollefsen $\text{et al}$.(J. Biol. Chem., $\text{257}$, 2162, 1982) and abilities of dextran sulfate and various glycosaminoglycans to activate the antithrombin activities of HC II and antithrombin III (AT III) were studied. By the purification method described here, highly purified HC II with the same specific activity as reported by Tollefsen $\text{et al}$. was obtained with a higher yield and in a shorter purification time. Heparin, dextran sulfate and chondroitin polysulfates 1 and 5 activated both HC II and AT III, while dermatan sulfate activated only HC II. Dextran sulfate was almost as active as heparin in the activation of HC II and AT III, indicating that in the interactions of heparin with HC II and AT III, sulfate groups of heparin are more important than carboxyl groups. When mixed with thrombin in the presence of dermatan sulfate, normal human plasma showed antithrombin activity which was not due to AT III but to HC II only. HC II did not inhibit factor Xa or plasmin in the presence of any glycosaminoglycans or dextran sulfate, suggesting that HC II would be a specific inhibitor of thrombin.     

A comparative study of low doses of heparin and a heparin analogue in the prevention of postoperative deep vein thrombosis

The efficacy of heparin and a semi-synthetic heparin analogue as compared for the prevention of postoperative deep vein thrombosis (DVT) in a prospective, randomized trial involving 200 patients. 12 (12.5%) out of 95 patients in the heparin group developed DVT compared to 6 (6.3%) out of 94 patients who received the analogue. Serious bleeding did not occur in any of the patients in either group and the difference in the operative and postoperative bleeding was not statistically significant. In this entire series of 190 patients, wound haematoma developed in 3, and all were in the heparin group. In 50 patients (25 in each group) plasma heparin levels, kaolincephalin clotting time (KCCT) and antithrombin III (At IIII) activity were measured in the samples withdrawn before, during and immediately after surgery and also on the first postoperative day. Significantly higher mean plasma heparin levels were obtained in the pat-ients receiving the analogue than those receiving heparin. Yet there was no difference in the prolongation of the KCCT observed in the two groups when measured by the clotting assay; the analogue had greater potentiating effect on At III activity as compared to heparin; the difference being statistically significant (p < 0.005). These findings provide further evidence for our preliminary observations that the heparin analogue selectively potentiates antithrombin III activity $\text{in vivo}$ while having little effect on overall clotting. The results presented indicate that it is as effective as heparin in preventing postoperative DVT.     

The inhibition of human plasma kallikrein by antithrombin III.

Prekallikrein, prekallikrein activator, high molecular weight kininogen and antithrombin III were isolated from human plasma. The prekallikrein was converted to kallikrein by prekallikrein activator. The kallikrein had arginine esterase activity and generated kinin from kininogen. Both activities were inhibited by antithrombin III in concentrations of approximately $\text{1}\text{2}$ of that of plasma. Physiological concentrations of heparin were required for the rapid and complete inhibition of kallikrein. In the absence of heparin only partial and much slower inhibition was achieved. The proteolytic, i.e. kinincleaving activity of kallikrein was more readily inhibited than the esterolytic activity.     

Radiolabelled ATIII as a probe for the detection of activation of blood coagulation invivo

Purified radiolabelled rabbit antithrombin III (ATIII) was used as a probe for $\text{in-vivo}$ activation of blood coagulation. Molecular weight distribution of radiolabelled ATIII and its complexes were studied $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vivo}$ by two techniques: gel exclusion chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. The radiolabelled purified ATIII readily formed radiolabelled complexes with activated procoagulants thrombin and factor Xa $\text{in}$$\text{vitro}$, as evidenced by SDS gel electrophoresis analysis. Gel exclusion chromatography separated ATIII from thrombin-ATIII complexes but not from Xa-ATIII complexes. The mean $\text{T}\text{-}\text{1}\text{2}$ of ATIII in rabbits was 42 hrs. The molecular weight distribution of infused radiolabelled antithrombin III was analyzed by gel exclusion chromatography and SDS gel electrophoresis. Circulating ¹²⁵I-labelled ATIII displayed an apparent molecular weight of 30,000 by gel exclusion chromatography as opposed to the apparent molecular weight of 68,000 for the purified protein. The gel chromatograms obtained at 10 minutes and 24 hrs displayed skewness toward higher molecular weight regions, suggesting complex formation of part of the circulating radiolabelled protein. Purified and circulating ATIII displayed identical molecular weights of 62,000 Daltons by SDS gel electrophoresis. Twenty-four hrs after $\text{in-vivo}$ administration, approximately 43% of ¹²⁵I-labelled ATIII displayed molecular weights greater than 62,000 Daltons by SDS gel electrophoresis, suggesting complex formation between ATIII and activated procoagulants under baseline physiological conditions. Thus, radiolabelled ATIII may represent a useful probe for the detection of physiological variations of $\text{in-vivo}$ thrombin generation.     

The in vivo increase of factor VIII activity by 2,3-diphosphoglycerate

When 2,3-Diphosphoglycerate (2,3-DPG) was injected intravenously into rats, factor VIII (antihemophilic factor) procoagulant activity was substantially increased. These effects are in accordance with what has been observed in hemolytic anemias and after intravenous injection of hemolysed blood. 2,3-DPG did not inhibit nor enhance any other clotting factor $\text{in vivo}$. $\text{In vitro}$ 2,3-DPG did not change factor VIII activity, indicating that the $\text{in vivo}$ increase of factor VIII, after 2,3-DPG injection, is not due to an activation, but rather to a $\text{de novo}$ release and/or synthesis or to a decrease in catabolism of factor VIII.     

Intravenous arachidonate in the mouse: A model for the evaluation of antithrombotic drugs

The intravenous administration of arachidonate to mice offers a rapid, convenient, and effective $\text{in}$$\text{vivo}$ model for the study of platelet aggregation and thrombosis, as well as the evaluation of potential antithrombotic drugs. Following intravenous administration in the mouse, there is dose-dependent cyanosis and respiratory distress with death at doses about 100 mg/kg. The rabbit is much more sensitive to the effects of intravenous arachidonate with death occurring at doses of 1 mg/kg; the rat shows an intermediate sensitivity. There is a relative specificity for the arachidonate effect: administration of certain other unsaturated fatty acids produces only minimal respiratory distress at equivalent doses. Respiratory distress in the mouse can be correlated with histologic evidence of platelet aggregation in the microvasculature of the lungs, but not in the heart or brain. Several nonsteroidal anti-inflammatory drugs (NSAID) which are known to be inhibitors of collagen-induced platelet aggregation and release, as well as inhibitors of prostaglandin synthetase, are able to block the effect of arachidonate $\text{in}$$\text{vivo}$. ADP inhibitors (dipyridamole, adenosine, and VK 744) are also effective in blocking the effects of arachidonate $\text{in}$$\text{vivo}$. Both the NSAID and the ADP inhibitors also block arachidonate-induced platelet aggregation $\text{in}$$\text{vitro}$.     

Molecular weight dependence of the anticoagulant properties of heparin: Intravenous and subcutaneous administration of fractionated heparins to man

A commercial mucosal heparin preparation has been fractionated by gel filtration into five different MW fractions of approximate mean MWs 11500–21500. These five heparins were randomly administered to 5 normal volunteers by intravenous and subcutaneous injection. The anticoagulant effects of the fractions were then determined by KCCT, anti-Xa clotting and anti-Xa chromogenic substrate assays. Following intravenous injection the high MW fraction produced identical elimination curves in all three assays, but with decreasing MW there was a divergence between results obtained by KCCT and anti-Xa assays. The lowest MW fraction produced between 2–3 times greater heparin levels when measured by anti-Xa assay. This divergence in assay results was produced as a consequence of the differences in specific activities seen when fractionated heparins were assayed in vitro by the different assay methods. That is, low MW heparin fractions had a higher anti-Xa specific activity than that determined by the KCCT assay. Conversely, high MW heparin had a greater specific activity when determined by the KCCT assay. When the response to intravenous heparin injection (measured by KCCT and anti-Xa assays) was compared to the dose administered it was found that the anti-Xa response was greater than expected. This increased plasma heparin like activity was not dependant upon the MW of the heparin fractions, and could not be neutralised by PF₄. A MW dependance was observed for the absorption of the heparin fractions into the circulation following subcutaneous injection, with low MW heparin producing higher heparin levels determined by all the assays.     

Monitoring of heparin and low molecular weight heparin with capillary and venous whole blood

A method for determination of antifactor Xa-like activity in capillary whole blood obtained from the fingertip is described, which employs the Heptest coagulation assay. Values obtained with capillary whole blood are compared to values of corresponding plasma and venous whole blood samples. Normal values in plasma, venous whole blood, and capillary blood from the fingertip were 17.1 +/- 2.1, 10.0 +/- 1.3 and 10.4 +/- 1.3 sec, respectively. The intraindividual coefficients of variations range from 0.4 to 1.8% in all assays. The day to day coefficient of variation of normal values ranged between 0.8 and 2.0% for all assays. The within assay coefficients of variation ranged from 3.0 to 7.7% for whole blood samples and from 1.5 to 2.2% for plasma samples after addition of no, 0.2 or 1.0 units of normal or LMW heparin to the samples. After administration of heparin or LMW heparin in healthy persons the coagulation values of the different coagulation assay systems displayed coefficients of correlation between r = 0.87 and r = 0.95. Correlation coefficients between the coagulation tests and the S 2222 chromogenic substrate method ranged from r = 0.77 to r = 0.92. In patients, who received LMW heparin for prophylaxis of thromboembolism the coagulation assay correlated between r = 0.78 and 0.92. The coagulation assays and the S 2222 method displayed coefficients of correlation between r = 0.74 and r = 0.83. The data indicate that Heptest sensitively measures antifactor Xa-like activity in capillary whole blood as well as venous whole blood samples containing low quantities of heparin or LMW heparin.     

Laboratory monitoring of continuous intravenous heparin therapy

The thrombolytic activity and elimination rate in vivo of a plasminogen activator purified from a melanoma cell line was examined in a rabbit thrombus model. Following an intravenous injection of ¹²⁵I labelled plasminogen activator, its biological activity disappeared very rapidly from the plasma ($\text{t}\text{1}\text{2}\text{= 1.5}\text{min}$) and radioactivity immediately accumulated in the liver. After a two-hour infusion with a total amount of 0.5 mg plasminogen activator a 60 per cent reduction in the weight of partially occluding thrombi was noted. No significant thrombolysis was seen in totally occluded vessels. Treatment with 0.5 mg plasminogen activator caused no depletion of fibrinogen or α₂-antiplasmin. No significant thrombolysis occurred after infusion of 0.5 mg streptokinase, but total lysis of occluding and non-occluding thrombi was obtained after a two-hour infusion of 1 mg streptokinase, without any reduction of fibrinogen. A marked reduction of α₂-antiplasmin was seen after both 0.5 and 1 mg of streptokinase. Haemorrhages were frequent during streptokinase treatment but were not seen in rabbits infused with plasminogen activator.     

The effect of heparin and dipyridamole on the deposition of fibrin-like material in rabbits infused with soluble fibrin monomer or fibrinogen

¹²⁵I-labelled fibrinogen or fibrin monomer was infused into rabbits treated with saline, heparin or dipyridamole. Serial measurements of radioactivity in blood were performed and the quantity of radioactivity excreted in the urine in 5 $\text{1}\text{2}$ hours was determined. The radioactive material deposited in various organs was measured and characterized. The results indicate that some fibrin-like deposition occurs in fibrinogen infused animals, the amount being unaffected by either heparin or dipyridamole. After FM-infusion, fibrin deposition especially in the spleen was substantially greater, the extent being inhibited by dipyridamole but not by heparin. It is concluded that the deposition of fibrin-like material in organs of animals infused with FM or fibrinogen may occur in the absence of thrombin elaboration. Alternative mechanisms of FM and fibrinogen polymerization are discussed.     

In vivo studies on the inhibition of coagulation by fractionated heparin and by a heparin analogue. II. Effects of a heparin analogue

SSHA, a semi-synthetic heparin analogue, and sodium heparin from porcine intestinal mucosa were injected subcutaneously into six healthy volunteers over a period of three days in a cross-over trial. Before injection and 2, 4, 6, 8 hrs afterwards, the heparin-like activity was measured with the APTT, the anti-Xa clotting test and two chromogenic substrate assays. The results show that SSHA mediates both anti-Xa and antithrombin activities in vivo. A comparison between the effects of SSHA and heparin is problematical due to the heterogeneity of different heparin preparations. Low doses of the analogue (45 mg s.c.) induced proportionally higher and longer lasting anti-Xa activities than higher doses (90 mg s.c.). Repeated injections of SSHA twice daily led to increasing effects on two tests for heparin-like activity, whereas two other tests remained unchanged. Both drugs were tolerated equally well, side effects were not detected. Clinical studies are required to demonstrate whether SSHA is similar or superior to low-dose heparin for use in thrombosis prophylaxis.     

An experimental model of in vivo fibrinolysis

An experimental model of $\text{in vivo}$ fibrinolysis was devised for estimation of $\text{in vivo}$ fibrinolytic activity in experimental animals (rat, rabbit and dog). The method consists of intravenous injection of homogenized human fibrin into an animal and subsequent immunological determination of fibrin degradation products (FDP) in a small amount of serum from the animal with the use of a commercial FDP kit for clinical use. In experiments with rats, FDP production was significantly increased by an injection of the fibrinolytic agent bisobrin while it was inhibited by treatment with the anti-fibrinolytic agent tranexamic acid (AMCA). These effects were in parallel with the effects of these agents on the euglobulin clot lysis time (ELT) determined in plasma samples from the treated rats. $\text{In vivo}$ fibrinogenolysis could also be measured by this method if human fibrinogen was used in place of human fibrin. Fibrinogenolysis was not observed in untreated rats, but it was observed in rats treated with bisobrin.     

A low molecular weight heparin compared with unfractionated heparin

A 6000 daltons low molecular weight heparin (LMWH) was compared with unfractionated mucosal heparin in vitro and in vivo. Despite unimpressive specifications by clotting assays in vitro, the LMWH gave high and sustained activity in vivo by anti-Factor Xa assays, following subcutaneous injection. However, activity measured by APTT and calcium thrombin time assays was at least as high as occurred following unfractionated heparin. On the basis of clotting assays, there seems no reason to expect a lower incidence of haemorrhagic side-effects following the clinical use of this LMWH. The study also strikingly demonstrates the inadequacy of in vitro clotting assays for assessing the in vivo behaviour of LMWH.     

A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, β-thromboglobulin and platelet factor 4 in healthy volunteers

A radioimmunoassay was developed for the platelet α-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml^?1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with β-thromboglobulin (β-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at ?20°C. Normal human plasma contained 105.0 ± 31.0 ng thrombospondin ml^?1 compared with β-TG concentrations of 37.2 ± 10.9 ng ml^?1 and PF4 concentrations of 14.7 ± 10.1 ng ml^?1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0–4°C. Release of thrombospondin during clotting of blood occurred at the same time as that of β-TG and PF4 and resulted in a serum concentration of 17.5 ± 5.5 μg ml^?1. Assay of whole blood gave a platelet thrombospondin content of 89.1 ± 28.3 ng/ 10⁶ platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml^?1, and was unrelated to urine flow. The half-life of thrombospondin $\text{in vivo}$ was about 9 h, much longer than that of either β-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.     

The release, distribution, and clearance of human beta-thromboglobulin and platelet factor 4.

β-Thromboglobulin (β-TG) is present in human plasma at 30.7 ± 13.7 ng ml^?1 (mean ± S.D.), approximately twice the concentration of platelet factor 4 (PF4). Plasma β-TG appears to equilibrate with synovial and amniotic fluids, but the PF4 concentration in these fluids is lower than in plasma. Neither protein passes freely into the cerebrospinal fluid. In each normal subject, the urinary concentration of β-TG is fairly constant at about 0.% of the plasma concentration. A higher percentage of immunologically intact PF4 enters the urine, but the concentration varies widely. β-TG added to the circulation by serum infusion is cleared with a $\text{1}\text{2}$-life of about 100 min., but the clearance of PF4 is so rapid that its $\text{1}\text{2}$-life cannot be estimated. The two proteins are released simultaneously from platelets $\text{in vitro}$. $\text{In vivo}$, the circulating concentration of PF4 is increased 20- to 30-fold by intravenous injection of 5000 units of mucous heparin, while β-TG levels remain unchanged.     

Elimination of intravenously administered radiolabelled antithrombin III and heparin in humans

Antithrombin III was purified from normal plasma by DEAE-Sephadex chromatography and heparin affinity chromatography; the protein was subsequently radiolabelled with 125I. 125I-antithrombin III alone and 125I-antithrombin III in the presence of high affinity 35S-heparin fractions were injected into normal humans. 125I-radiolabel and protein bound 35S-radioactivity were followed separately. In semilogarithmic plots 125I-antithrombin III disappeared according to a double exponential curve with a half-life in the second phase of 56.8 hr in the absence of heparin and of 33.7 hr in the presence of heparin. Protein bound 35S-radioactivity disappeared much faster than the 125I-radiolabel. These data support the concept that heparin disappears as free heparin from the equilibrium heparin - antithrombin III in equilibrium heparin + antithrombin III. Immuno-reactive antithrombin III decreased from 100% to 85-90% immediately after injection of 125I-antithrombin III in the presence of heparin and returned to normal values within 30 min. This suggests that antithrombin III is transiently sequestered, possibly in trimolecular complexes consisting of antithrombin III, heparin and either lipases or other vascular bound proteins.     

Effects of standard heparin and a low molecular weight heparin (Kabi 2165) on fibrinolysis

Previous and recent reports have suggested a fibrinolysis-enhancing property of standard heparin and low molecular weight heparins, but these observations have never been confirmed in a study fulfilling appropriate methodological criteria. The aim of this study was to evaluate the effect of standard heparin and a low molecular weight heparin (Kabi 2165) on fibrinolysis in a randomized cross-over double blind placebo controlled study. Six healthy volunteers received intravenously a bolus dose of the following treatments: placebo; standard heparin, 5,000 I.U.; Kabi 2165, 5,000 anti-Xa U; Kabi 2165, 10,000 anti-Xa U. Before the injection and at established times thereafter, blood samples were collected for the following assays in plasma: t-PA activity, PA inhibitor activity, fibrin plate lysis area (FPLA), plasminogen, alpha 2-antiplasmin, fibrinogen and anti-Xa activity. Placebo and Kabi 2165, 5,000 anti-Xa U, had no effect on t-PA plasma level. Standard heparin and Kabi 2165, 10,000 anti-Xa U, produced a statistically significant increase in t-PA level at 1 hour after the infusion. This increase lasted for at least 1 hour after the infusion. No effect of any treatment on PA inhibitor, plasminogen, FLPA, alpha 2-antiplasmin and fibrinogen was observed. We conclude that an intravenous bolus dose of both standard heparin, 5,000 I.U. and Kabi 2165, 10,000 anti-Xa U produces a delayed and sustained increase in plasma t-PA.