anticoagulant effects of sulphonated polyurethanes

339

~~coa~lant polyurethanes

effects of sulphonated

James H. Silver, Arlene P, Hart, Eliot C, Williams* and Stuart L. Cooper departments of Chemical Engineering and Medicines, Univefsf~ of Wisconsin-Madison, 1415 Johnson Drive, Madison, WI 53706, USA

Said Charef, Denis Labarre and Marcel Jozefowicz Laboratoire de Recherches sur /es Macromolecules, Universit& Paris-Nerd, Avenue J.6. Clement, 93430, Villetaneuse, France

Sulphonated polyurethanes have been shown to have excellent blood contacting properties. In this paper, similar polyurethanes which are water soluble have been investigated to determine their influence on thrombus formation. These polymers were shown to delay clotting times in the following ways: by direct complex formation between the polymer and thrombin; by interference with fibrin polymerization; and by complex interactions between polymer, thrombin, plasma antiproteases and fibrinogen in plasma.

Keywords: Blood-material interactions, polyurethanes, sulphonates

Received 16 April 1991; revised 17 July 1991; accepted 18 July 1991

To prevent blood coagulation at the surface of synthetic biomaterials, a number of investigators have bound heparin, a natural anticoagulant, to these surfaces*. Heparin is a mucopolysac~haride which inhibits blood coagulation by promoting the activity of serine protease inhibitors, primarily antithrombin III, to block the coagulation enzymes thrombin’, 3, factor Xa, and to some extent, factors IXa, XIa, and XIIa3. However, when heparin is ionically bound to surfaces, its anticoagulant effects are related to its release in to the bloodstream. For long-term applications, this strategy will fail, because eventually the heparin is depleted. Other investigators have covalently attached heparin to biomaterial surfaces, and have sometimes shown a decrease in the thrombogenicity of these surfaces. This approach may be of limited use, as heparitinases eventually degrade the heparin’. To this end, modification of biomaterials by incorporation of ionic functional groups, similar to those which endow heparin with its anticoagulant properties, has been attempted by many investigators.

Jozefowicz et al.4-6 have described modification of dextrans and polystyrene by incorporation of sulphonate and carboxylate moieties to mimic the anticoagulant activity of heparin. We are interested in ionic modification of polyurethanes, since these materials have such good physical properties, and because they can be chemically tailored to meet other challenges which occur in biological environments7. Previous work by Grasel’ has demon-

Correspondence to Professor S.L. Cooper

strated that polyurethanes which are modified by grafting propyl sulphonate groups onto the urethane nitrogens have extremely good blood contacting properties in a canine ex viva shunt model. Observation by scanning electron microscopy showed that very few platelets adhered to these materials, and those which did adhere did not undergo shape change. However, in these same experiments, significant amounts of fibrinogen were adsorbed. Although adsorption of fibrinogen is usually associated with thrombogenicity, this may not be true of sulphonated polyurethanes.

In this paper, the anticoagulant properties of polyurethanes similar to those examined by Grasel were investigated. The ionic content of the polyurethanes was sufficient to make them water-soluble. We measured the ability of these polymers to inhibit thrombin activity, either by directly binding thrombin, or by catalysing inhibition of thrombin by serine protease inhibitors (heparin-like effects), or by interfering with polymerization of fibrinogen in plasma.

Anticoagulant activity was demonstrated by measuring the clotting times of solutions of polymer in human plasma, or purified fibrinogen solutions, to which thrombin was added. Interference with the intrinsic or extrinsic pathways of the coagulation cascade was determined using activated partial thromboplastin time tests, or prothrombin time tests, respectively. To determine whether the increase in clotting times resulted from thrombin inhibition, or from alteration of the clotting properties of fibrinogen in plasma, clotting times were

D 1992 Butterworth-Heinemann Ltd 0142-9612/92/060339-06

Biomaterials 1992, Vol. 13 No. 6

340 Anticoagulant effects of sulphonated polyurethanes: J.H. Silver et al.

measured when thromhin was replaced by the snake Vi&urn) was purchased from Helena Labs (Beaumont, venom enzyme, reptilase. TX, USA).

Interference with fibrin formation was demonstrated by adding polymer to mixtures of purified thrombin and fibrinogen, in the presence of factor XIII. When these samples were subjected to electrophoresis under denaturing and reducing conditions, there was evidence of cleavage of both fibrinopeptides A and B, but not of fibrinogen gamma chain ligation, indicating that fibrinogen was being converted into fibrin but not polymerizing to fibrilsg. A direct effect of polymer on fibrin polymerization was demonstrated by preparing solutions of fibrin monomer”, and allowing them to polymerize in the presence of polymer,

Coagulation tests

Interactions between polymer and thrombin were also assessed by using a thrombin-specific chromogenic substrate. In these experiments, thrombin activity was measured in the presence of polymer alone, or polymer and plasma. Studies of the interaction between polymer, thrombin, and antiprotease will be included in a subsequent paper.

Thrombin times [TT) and reptilase times [RT) PEU-SO,-0.40, 100 ~1, at concentrations ranging from 0 to 3 g/l in Michaelis buffer (26 mM sodium barbital, 26 mM sodium acetate, 0.1 M NaCl, pH 7.3) was incubated with 200 ,a1 human PPP, for 15 min at 37’C. Then 100 ~1 human thrombin was added (final concentration 7.5 NIH units/ml), and the clotting time determined using a fibrometer (BBL Fibrosystem, Beckton Dickinson, Parisippany, NJ, USA). Control studies were performed in which the polymer was replaced with buffer, and the level of thrombin was varied. These experiments were repeated in antithrombin III-depleted plasma, or in fibrinogen (4 g/l) solution containing BSA (30 g/l). Tests in both PPP and fibrinogen solution were repeated using reptilase in place of thrombin.

Pro throm bin times (PT) EXPERIMENTAL

Materials

The base polyurethane was synthesized by reaction of methylene bislp-phenyl isocyanate), l,Cbutane diol, and polytetramethylene oxide in a 3/2/l mole ratio. Derivatives of this material were made by replacement of 30,40 and 80 mol% of the urethane hydrogens with propyl sulphonate groups’. PEU refers to the nonionized polyurethane: PEU-SC&-XX refers to the derivative of the polyurethane, with XX being the mole fraction of urethane hydrogens replaced by propyl sulphonate groups.

Either PEU-SO,-0.30 or PEU-SO,-0.40, 0.2 ml, at 1.5 mgfml in phosphate buffered saline at pH 7.35 (PBS) was added to 0.2 ml fresh frozen canine PPP. PEU-SO,- 0.80,O.Z ml, at 2.0 mg/ml in tris-buffered saline solution, pH 7,4 (TBS) was also added to 0.2 ml fresh frozen canine PPP. The polymer-plasma mixture and 0.2 ml thromboplastin were warmed separately for 5 min at 37°C. Then the thromboplastin was added to 0.1 ml plasma mix, and the clotting time recorded using the Fibrosystem fibrometer.

Proteins

Human and canine titrated platelet poor plasma (PPP) were obtained from volunteer donors by drawing 9 volumes of blood into 1 volume of 3.8% trisodium citrate, and then centrifuging at 5OOOg. Antith~mbin III-depleted human plasma (PPP-AT) was prepared from fresh PPP by affinity chromatography on an anti-antithrombin III Sepharose column as previously describedll. Residual antith~mbin III was <3.5% of normal, as assessed in the presence of heparin and bovine thrombin, using a chromogenic substrate, Human fibrinogen was purchased from Sigma Chemical (St. Louis, MO, USA), and was >97% clottable, Human thrombin (2.6 mglml, 4035 NIH units/ml) was the generous gift of Dr J.W. Fenton II, Human thrombin was also purchased from Diagnostica Stago (France). Bovine thrombin was purchased from Armour Pharmaceutical Co. (Kankakee, IL, USA), and used without further purification. Bovine serum albumin (BSA) was purchased from Sigma (St, Louis, MO, USA), and used as received. Reptilase was obtained from Diagnostica Stago (France). Plasma factor XIII was the generous gift of Dr Deane Masher. Thromboplastin and cephaloplastin were purchased from Baxter Scientific Products (McGraw Park, IL, USA). Purity was determined by electrophoresis, and all proteins were snap frozen in an ethanol-dry ice bath, and stored at -70°C until use, Thrombin-specific chromogenic substrate, S-2238, (Kabi

Act&a ted partial throm boplastin times (APTT] Both the polymer-plasma mix, as described for the prothrombin test, and activated cephaloplastin were prewarmed to 37°C for 5 min; 0.1 ml of each solution was added to 0.1 ml CaCl, (0.02 M), and the clotting time recorded, again using the Fibrosystem fibrometer.

interference with fibrin cross-~jn~jffg To plastic test tubes, the following were added in order: 0.6 ml of tris buffer (TBS, pH 7.4,0.15 M NaCl), 0.1 ml of CaCl, (0.02 M), 0.1 ml polymer (1.0 g/l) or buffer or heparln (100 USP units/ml)~ 0.1 ml fib~nogen (2.15 mglml), and 50~~1 human thrombin (106.5 NIH units/ml). The reaction was allowed to proceed for at least 2 h at 37% Next, 0.5 ml of 6 M urea, 3% sodium dodecyl sulphate, 3% P-mercaptoethanol was added to each sample overnight at 37%. These samples were then applied to 8% polyacrylamide gels, and electrophoresis was performed for 8 h at 45 mA, Protein bands were detected by silver staining. These experiments were repeated in the presence of Z- and 20-fold molar excess of calcium, with respect to the sulphonate content of the polyurethanes. Plasma factor XIII was also added to the system containing PEU-SO,-0.40, fibrinogen, and thrombin, and final concentrations were 10 mM calcium, and 1, 3, 10, and 30 pglrnl of exogenous plasma factor XIII in excess of any residual factor XIII which was present along with the fibrinogen,

Using a different method to demonstrate interference with fibrin assembly, fibrin monomer (des-AABB fibrinogen) was prepared in 1 M NaBr, 0.05 M sodium


Anticoagulant effects of sulphonated polyurethanes: J.H. Silver et ai. 341

Table 1 Clotting times.

Polymer Concentration Thrombin time (m9iml) (s)

Activated partial thromboplastin time (a)

Prothrombin time ts)

PEU-SO,-0.30 0.15 53 rf:16 >200 71 f21 PEU-SO,-0.40 0.15 51 k 9 >200 79 2 25 Polyvinyl alcohol 0.20 21.6 + 0.4 19.0 IL 0.4 23.2 rt 0.8 Phosphate buffered saline - 23.9 rt 0.4 17.7 IL 0.4 23.8 rt 0.6

Values are an average of two determinations

acetate, pH 5.3, at a concentration of 8.2 mg/ml, as described by Rampling”‘. Fibrin monomer, 30 p 1, 240 .u 1 phosphate buffer (pH 6) and 30~1 of either PEU-SO~~.30 (final concentration 0.15 mglml) or PEU-SOB-0.40 (final concentration 0.15 mglml) or PEU-SO,-0.80 [final concentration 0.10 mg/ml) or polyvinyl alcohol (PVA) (final concentration 0.2 mg/ml); or buffer, were added to a glass test tube in a 37’C water bath. The solution was swirled to mix, returned to the 37°C water bath, and then personally observed for formation of fibrin strands or a solid gel.

Direct thrombin inhibition by PEU-SO,-0.40 The polymer was diluted in buffer containing 50 mM trisHC1, 0.1 M NaCl, bovine serum albumin (0.5 g/l), and EDTA d&odium salt 2.8 g/l at pH 6.4. A similar buffer, but without EDTA, was used for dilutions of human or bovine thrombin. Polymer solution, 200 ~1, at increasing concentrations from 0 to 2 mg/ml was incubated with 200~1 of thrombin (3 NIH units/ml) for 1 min at 37°C. Then 2OO~.rl S-2236 was added and the absorbance at 405 nm was recorded as a function of time at 37°C on a Perkin-Elmer Lambda 4C UV-Vis spectrophotometer, for 1 min. The residual thrombin activity was determined by comparison with a calibration curve, established in a similar experiment, in which polymer was replaced with buffer, and the thrombin concentration varied.

RESULTS

In the presence of PEU-SO,-0.40, the clotting times of PPP were prolonged in TT, PT, and APTT tests (Table 2 ), Clotting times in TT, PT, and APTT tests were also prolonged in the presence of PEU-SO,-0.30, as compared to controls containing PVA or buffer (Table 1). Reptilase times remained similar to controls without polymer (Table Z), for polymer concentrations of 0.75 mg/ml or less. In contrast, at higher polymer concentrations, reptilase times were prolonged in normal human plasma (data not shown). InFigure 2, the thrombin time is plotted against the amount of polymer present in solution. This

Table 2 Clotting times for PEU-SO,-0.40.

Polymer Plasma + Fibrinogen + Fibrinogen + concentration reptitase reptiiase thrombin (m9/ml) (s) (s) (s)

0 30 * 2 30 k 2 6 F 0.3 0.25 30 f 2 >45 15 f 0.8 0.50 30 f 2 >45 18 + 0.9 0.75 30 * 2 >45 25 + 1.9

Values are an average of at least two determinations

2.5

I I 1 I , IO 0.0 0.2 0.4 0.6 1.0

Polymer concentration [mglml)

Figurei Inhibition of thrombin by PEU-SO,-0.40 in PPP (open symbols) or in PPP-AT (solid symbols) assessed in thrombin time tests. The thrombin times are indicated by triangles, and the calculated amount of residual thrombin is indicated by circles.

figure also indicates the amount of thrombin which would have to be present to give similar clotting times. The residual thrombin in this system decreased linearly with the amount of polymer present in solution, up to a final polymer concentration of about 0.25 mg/ml [Figure 2). In antithrombin III-depleted plasma, the thrombin activity observed to be inhibited was roughly half of that observed in normal plasma. When PPP was replaced by fibrinogen-albumin solution, the clotting times were prolonged when initiated by either thrombin or reptilase (Table 2). Since this result suggested some interference with fibrin polymerization, the amount of thrombin inhibited in this system could not be calculated.

The hypothesis that these materials directly interfere with the polymerization of fibrin was tested electro- phoretically. All samples contained factor XIII, since fibrinogen was not treated to remove this zymogen’z. Controls of clotted and non-clotted, reduced fibrinogen are shown in lanes 6 and 7, respectively, of Figure 2. The non-clotted control sample shows a band at a molecular weight of 46.5 X 103, which is the y-chain of fibrinogen. This band is reduced in intensity in the clotted control sample, and a new band appears at molecular weight 93 X 103, due to ligation of gamma-chains of neighbouring fibrin monomers by factor XIIIa to form y-y dimersg. Further, the Au-band and BP-band in the non-clotted, reduced fibrinogen sample (lane 7) are distinguishable from the a-bands and P-bands, which are seen in all of

Biomaterials 1992. Vol. 13 No. 6

342 Anticoagulant effects of suiphonated polyurethanes: J.H. Silver et a/.

93

36

Y-Y

Aa a

1 2 3 4 5 6 7

Figure 2 SDS-PAGE of fibrinogen and thrombin, reacted in the presence of: heparin (lane 1); PVA (lane 2); PEU-S0,0.80 (lane 3); PEU-SO,-0.40 (lane 4); PEU-SO,-0.30 (lane 5); buffer (lane 6). Non-clotted, reduced fibrinogen is shown in lane 7. All samples were run under reducing conditions.

the other samples. This indicates that thrombin effectively cleaves fibrinopeptides A and B from fibrinogen under the test conditions. Formation of y-y dimer was inhibited in the presence of PEU-SO,-0.80, PEWSO,-0.40, and PEU-SOL-0.30, as seen in lanes 3,4 and 5, respectively, of Figure 2. The absence of the 93 000 molecular weight band in these cases provides evidence that protofibril formation is being inhibited. Further, in the presence of the sulphonated polymers, thrombin can be seen as a distinct band at 36 000 molecularweight in lanes 3,4, and 5. Normally, thrombin binds to fibrin clots’3, and so the thrombin band is less noticeable in the control lanes. The appearance of thrombin as a distinct band suggests that these sulphonated polyurethanes must bind to either thrombin or fibrin, preventing them from binding to each other, after cleavage of the fibrinopeptides. Heparin and PVA were used as controls for excluded volume, viscosity, and charge effects, and are shown in lanes 1 and 2, respectively. Neither PVA nor heparin has been reported to have any direct effect on the formation of protofibrils, and neither was seen to have any effect under these conditions. Clotting activity for the samples containing PEU-SO,-0.30 or PEU-SO,-0.40 was not restored in the presence of excess calcium, which demonstrates that fibrin formation was not being inhibited via simple titration of available calcium by the polymer [results not shown). Excess activated plasma factor XIII also failed to re-establish cross-linking of gamma-chains of fibrinogen in these samples (results not shown).

PEU-SOB-0.30 and PELT-SO,-0.40 were added to solutions of fibrin monomer at its pl, and the monomer was allowed to polymerize by raising the solution pH over its p1. This permits electrostatic forces to assemble protofibrils. In the presence of PEU-SO,-0.30 and PEU-SO,-0.40, neither fibrin strands nor a solid fibrin gel were detected at up to 20 h of observation. Instead, white clumps on the order of 1 mm diameter formed immediately, and presumably contain a complex of polymer and fibrin monomer. In contrast, controls containing either polyvinyl alcohol (PVA) or buffer polymerized normally (Table 3).

Table 3 Fibrin monomer clotting times.

Polymer Concentra- Formation of Formation of tion weak fibrin solid fibrin (mQ/mf) strands (min) gel (min)

PEU-S03-0.80 0.10 None detected None detected PEU-SO,-0.40 0.15 None detected None detected PEU-SO3-0.30 0.15 None detected None detected Polyvinyl 0.20 10 + 0.5 20 + 1 alcohol Phosphate - 9.5 + 0.5 20 + 1 buffered saline

Values are an average of two determinations

In the presence of the PEU-SO,-0.40, human or bovine thrombin activity assessed by lysis of chromogenic substrate (S-2238) decreased with increasing polymer concentration (Figure 3): 0.2 mg of polymer was able to inhibit roughly one fourth of the thrombin initially present. Thus, the polymer exerts its anticoagulant effects at least partially though inactivation of th~mbin. Since thrombin contains an anion-binding exosite through which it binds to heparin14*15, it would not be too surprising to find some complexation between the anionomer and the protease,

In plasma systems, the anticoagulant effect of these polymers seems to be more potent than in single- component solutions of thrombin. As shown inFigure 3, the equivalent of 21 NIH units of thrombin were inhibited in normal plasma, per milligram of polymer. Roughly half of this amount of thrombin was inhibited in antithrombin III-depleted plasma. In comparison, 1 NIH unit of thrombin was directly inhibited, per milligram of PEU-SO,-0.40, as shown in Figure 3.

DISCUSSION AND CONCLUSIONS

These results provide some insight into the mechanisms by which sulphonated polyurethanes exhibit their


Anticoagulant effects of sulphonated polyurethanes: J.H. Silver et al. 343

.- A

.Q E 2

l

5 0.4 - l

0 . 0

2 1- 3 0.2 - GYt

0

I I I t I I

0.2 0.4 0.6 0.8

Polymer concentration (mglmll

Figure3 Direct inhibition of human thrombin (circles) or bovine thrombin (triangtes) by PEU-SO,-0.40, as assayed by lysis of chromogenic substrate (S-2238).

excellent blood-contacting properties, observed previously’. These polymers were capable of directly inhibiting roughly 1 NIH unit of thrombin activity per milligram of polymer. In the more complicated plasma milieu, their apparent thrombin inhibitory activity was increased to 21 NIH units per milligram in normal human plasma, and roughly half that in antithrombin III- depleted plasma. This suggests that the polymer may be interacting with antithrombin III in a heparin-like manner. It is also possible that the polymer and antithrombin If1 act in independent ways to prolong clotting times. These possibilities are currently being investigated. Since prolongation of clotting times was observed in the TT, PT, and APTT tests, the polymers must inhibit coagulation beyond the point where the intrinsic and extrinsic pathways overlap. Prolonged reptilase times in fibrinogen-albumin solutions indicate that the clotting properties of fibrinogen are altered in the presence of PEU-SO,-0.40. Similar results are observed in normal human plasma, at polymer concentrations greater than 0.75 mg/ml. Reptilase causes fibrinogen to clot by cleavage of fibrinopeptide A (FPA) only, and not fibrinopeptide B (FPB)‘“,

These results can be compared to studies on randomly sulphonated and carboxylated dextran derivatives by Jozefonvicz et aL6, 17. On their more active compounds, they found prolongation of TT, PT, APTT, and a null effect on reptilase times, While the results here are qualitatively similar, they differ in detail, partially due to the differences in the conditions used. In addition, the modified polyurethanes in this work are multiblock copolymers. Thus, while the sulphonate derivation is random cm the urethane nitrogen functions, the overall placement of the urethane functions is decidedly non- random along the backbone of the polymer.

Inhibition of cross-linking of fibrin, as shown in Figure 2, indicates that the polymer must either complex free calcium, inhibit thrombin, inhibit factor XIIIa, or alter the clotting properties of fibrin. By addition of excess reagent, it was demonstrated that the polymer

-

was not simply complexing free calcium, nor interfering with factor XIIIa. Thrombin activity in the electrophoretic study was not completely inhibited, since cleavage of fibrinopeptides A and B occurred. Thus, these polymers directly inhibit fibrin assembly.

Further proof of this is evident from the fact that in solutions of fibrin monomer and PEU-SO,-0.30 or PEU-SO,-0.40, the fibrin monomer complexes with the polyurethane and therefore cannot properly align to form protofibrils. In fibrin monomer, after cleavage of both FPA and FPB, the central globular domain, or E domain, is positively charged” at physiological pH. Thus, electrostatic interaction with the anionomer may be responsible for aggregate formation. This would explain the prolongation of clotting times seen in T&e 3. Control studies using PVA did not show prolongation of clotting times in fibrin monomer solution, as seen in Table 3. Further, reptilase times would be less sensitive to electrostatic effects at lower concentrations of polymer, as observed in normal human plasma.

It is of interest to consider the relevance of these observations on the soluble sulphonated polyurethanes to the ex viva results on related polymers of lower degrees of sulphonation” 1’S “, These polymers are insoluble in aqueous media, but are highly swollen. Ex viva, they exhibit low extents of thrombus deposition and platelet activation despite high levels of fibrinogen deposition. The antithrombogenic effects of these swollen sulphonated polyurethanes may be due, in part, to inhibition of thrombin activity. They may also have an effect on fibrin assembly. However, preadsorption of canine fibrinogen onto these surfaces before blood contact in a canine ex vjvo model, exhibits a very th~mbogenic response (results not shown). This suggests that the anticoagulant and fibrinogen interaction effects of the soluble polymers studied here may not be of primary importance in the antithrombogenic mechanisms of this material, Interestingly in situations involving high flow rates, Jozefonvicz et a1,““7, have found that the anticoagulant properties of s~lphonated polysaccharide resins are qualitatively similar to soluble versions of the same material.

Strong interactions between sulphonated polyurethanes and fibrinogen were also suggested by the work of Santerre et al.“, who demonstrated that fibrinogen could not be displaced from such surfaces in Vroman effect experiments, except at very long times. Early investigation?, ” have shown that dextran sulphates precipitate fibrinogen,

In conclusion, we have demonstrated that sulphonated polyurethanes prolong clotting times in TT, PT and APTT tests. These polymers act to directly inhibit thrombin activity, as demonstrated using ct chromogenic assay. However, its anticoagulant effect is more potent in systems containing plasma or fibrinogen than could be explained by inhibition of thrombin alone. We demonstrated that fibrinmonomer aggregates in the presence of these suIphonated polyurethanes, and thus cannot properly align to form protofibrils, Finally, it appears that these sulphonated polyurethanes have complex interactions with thrombin, fibrinogen, and plasma antiproteases. Whether these soluble materials behave in the same manner as solid surfaces of similar chemistry remains to be determined.


Anticoaaulant effects of sulehonated Dolvurethanes: J.H. Silver et al. . _

The authors would like to acknowledge the many helpful discussions with Dr Deane Mosher (Department of Hematology, University of Wisconsin-Madison, USA) and with Drs Anne-Marie Fischer and Jacqueline Tapon- Bretaudiere (HBpital Necker, Paris). This work was supported in part by the National Institutes of Health, through Grants HL-21001 and HL-24046.

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