Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors

Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors

Jovil Kannampuzha, Anatharam Kalya, and Alexandra Lucas

Abstract

In the human body, an intricate mechanism has evolved to limit blood loss from damaged blood vessels through formation of clot, while maintaining blood in a fluid state, such that the circulation remains intact. This system is required for maintaining the integrity of the circulatory system as perturbations in the balance between procoagulant and anticoagulant forces can lead to bleeding or thrombotic disorders. Formation of the key coagulation enzyme, the serine protease thrombin, proceeds through a tightly regulated series of reactions involving a cascade of activation of plasma proteases and cofactors. The steps in the serine protease activation cascade are regulated by inhibitors, termed serpins, also called serine protease inhibitors. In order to define serpin functions, it is also necessary to understand their target proteases. One of the central protease pathways is the thrombotic or clot-forming pathway. The tissue factor and contact activation pathways both activate the final common pathway of factor X, thrombin, and fibrin. In this chapter we define the basic clinical methods used to measure factor X (FX).

Keywords Serine protease, Factor X, Thrombosis, Thrombolysis

1 Introduction

Formation of a fibrin clot is mediated by a group of tightly regulated plasma proteases and cofactors. While this system is essential for minimizing blood loss from an injured blood vessel (hemostasis), it also contributes to pathologic fibrin formation and platelet activation that may occlude vessels (thrombosis). The responsibil- ity of the clotting cascade is to rapidly convert liquid blood into a stable colloid state at the site of vascular damage, a colloid designed to restrict further blood loss. The two major pathways involved in initiating the clotting cascade are the tissue factor (TF) pathway (also known as the extrinsic pathway) and the “contact” pathway

Alexandra Lucas (ed.), Serpins: Methods and Protocols, Methods in Molecular Biology, vol. 1826, https://doi.org/10.1007/978-1-4939-8645-3, © Springer Science+Business Media, LLC, part of Springer Nature 2018 267 268 Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors

(or intrinsic pathway). The TF pathway is primarily responsible for normal hemostasis, while the contact pathway is thought to be a major contributor to thrombosis (a pathologic process) [1]. However, both pathways can contribute to either normal hemostasis or also to excess thrombosis and both eventually converge at factor X (FX) and thrombin (FII). Of interest the initiating or central TF pathway begins by activation of a non-serine protease, TF which is a cellular (often platelet) receptor, which then activates the sequentially activated serine protease pathways. Together the balance between thrombotic (clot formation) and thrombolytic (clot breakdown) pathways are maintained and the serine proteases in both pathways are regulated by serpins. Treatment with heparin is known to activate antithrombin III (ATIII; SERPINC1), the first reported serpin target used for ther- apeutic purposes and, through the wide use of heparin, still one of the most commonly targeted antithrombotic agents.

1.1 TF and Activation Endothelial damage exposes platelets to vascular collagen and of the Coagulation, plasma factor VII/VIIa (FVII/FVIIa) to extravascular TF. While Serine Protease, central to activation of the clotting cascades, the sequentially acti- Pathways vated serine proteases, TF is not itself a serine protease but rather a receptor usually found on the platelet membrane surface. TF acts as the initiating nidus for coagulation pathway activation. Proteins, such as von Willebrand factor (vWF), facilitate the binding of platelets to the injured vessel wall. This TF:FVIIa complex is pro- posed to be the primary activator of the coagulation protease cascade in vivo. TF is the cell surface, predominantly the platelet surface, receptor for factor VII, a serine protease. The main role of the TF pathway is to generate a process by which thrombin is released/ activated very rapidly due to its importance in feedback activation roles within the cascade. With this brief chapter, we describe the clinical laboratory assay for FX, one of the central serine protease mediators of coagulation (see Fig. 1). The process includes the following steps (Fig. 1): 1. Following damage to the blood vessel, FVII (which circulates in a higher amount than any other activated coagulation factor) leaves the circulation and comes into contact with TF expressed on tissue factor-bearing cells (platelets, stromal fibroblasts, and leukocytes), forming an activated complex (TF-FVIIa). 2. TF-FVIIa activates FIX and FX. 3. FVII is itself activated by thrombin, FXIa, FXII, and FXa. 4. The activation of FX (to form FXa) by TF-FVIIa is almost immediately inhibited by tissue factor pathway inhibitor (TFPI). 5. FXa and its cofactor FVa form the prothrombinase complex, which activates prothrombin to thrombin. Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors 269

Fig. 1 Serine protease clotting cascade is initiated by tissue factor (TF) and factor VII (VII) activation which is now believed to begin protease cascade activation of clotting factors in what is referred to as the TF and contact, or the classic extrinsic and intrinsic pathways, respectively. Anti-thrombin III (ATIII) inhibits thrombin and factor X. ATIII has been used clinically via heparin activation as the earliest serpin therapy

6. Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which forms a complex with FIX), and activates and releases FVIII from being bound to vWF. 7. FVIIIa is the cofactor of FIXa, and together they form the “tenase” complex, which activates FX; and so, the cycle continues.

1.2 Tissue Factor The assays looking at coagulation pathways follow a similar prin- Assays, the Extrinsic ciple. Plasma deficient in any of the factors in the TF pathway will Cascade result in a prolonged prothrombin time (PT). Factor-deficient plasma can be used in the identification and quantification of the deficient factor in the patient’s plasma. Decreased levels of factor II, V, VII, or X may be found in congenital deficiency of each factor or may be acquired secondary to other diseases such as liver disease, disseminated intravascular coagulation (DIC), or due to specific inhibitory proteins. Antibodies that inhibit the activity of a specific coagulation factor can develop spontaneously or in associa- tion with certain medications, autoimmune diseases, or other con- ditions. These antibodies may also arise when a patient with a hereditary factor deficiency is transfused with a product containing the factor, such as a factor concentrate or fresh frozen plasma. These antibodies can also affect the accuracy of these assays and should be considered. 270 Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors

1.3 Thrombin or In this “intrinsic pathway,” coagulation may be initiated when fac- Contact Pathway, tor XII is activated on a charged surface by a process called contact the Intrinsic Pathway activation [2]. Activation of factor XII is followed sequentially by activation of factor XI and factor IX. The intrinsic and extrinsic pathways converge at the level of FX activation. Factor Xa activates prothrombin to thrombin in the presence of the cofactor factor Va, and thrombin subsequently converts fibrinogen to fibrin [3]. FX and thrombin are inhibited by ATIII (Fig. 1), a serpin that is activated 100–1000-fold by heparin.

1.4 Contact Pathway The intrinsic pathway is typically depicted as a sequence of proteo- Assays lytic reactions culminating in factor IX activation. Plasma deficient in any of the factors in the intrinsic pathway will result in a prolonged activated partial thromboplastin time (aPTT). Here too, factor-deficient plasma can be used in the identification and quantification of the factor deficient in the patient’s plasma. A mixture of the deficient plasma and the patient plasma is tested by aPTT, and the result is interpreted using a reference curve obtained with dilutions of calibration plasma or a normal plasma pool mixed with the deficient plasma. The percent of factor activity present in the plasma can be determined by the degree of correction obtained with the aPTT when the test plasma is added to specific factor- deficient plasma. Decreased levels of factor VIII or IX may be found in congenital deficiency of each of these factors resulting in hemophilia (FVIII deficiency) or Christmas disease (factor IX deficiency) or may be acquired secondary to other diseases, similar to what is seen in extrinsic pathway abnormalities. Human factor X (FX) is a two-chain, vitamin K-dependent, plasma glycoprotein which is synthesized in the liver. During coagulation, FX is proteo- lytically activated to the serine protease, factor Xa, by the intrinsic factor Xase complex (factor IXa, factor VIIIa, cellular surface, and calcium ions). Factor Xa, combined with calcium, factor Va and the negatively charged phospholipid surface, forms the prothrombinase complex that is responsible for the rapid conversion of prothrombin to thrombin. Patients receiving oral anticoagulant therapy or with a vitamin K deficiency due to intake or absorption abnormalities will have reduced plasma levels of FX, a vitamin K-dependent clotting factor. Factor X activity in a patient’s plasma is determined by performing a modified prothrombin time test (PT). Patient plasma is diluted and added to a plasma deficient in factor X. Correction of the clotting time of the deficient plasma is proportional to the concentration (% activity) of that factor in the patient plasma, interpolated from a calibration curve. In essence, plasma deficient in any of the factors in the extrinsic pathway will result in a prolonged PT [4, 5]. Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors 271

2 Materials

2.1 Factor X 1. ACL®Top 500 CTS Coagulation Analyzer (a hemostasis test- Assay ing system). 2. Cuvettes. 3. ACL®Top specimen racks. 4. HemosIL calibration plasma (only needed if calibrating). 5. HemosIL factor diluent. 6. HemosIL® RecombiPlasTin 2G (20 mL) contains lyophilized reagent and an aqueous diluent. 7. HemosIL Factor X Deficient Plasma is lyophilized human plasma, artificially depleted of FX, and containing buffer and stabilizers. Residual FX activity <1% and other factors have normal levels.

3 Methods

3.1 Preparation 1. RecombiPlasTin 2G: Allow each vial of reagent and diluent to equilibrate at 15–25 °C for at least 15 min before reconstitution. 2. Pipette the exact amount required (20 mL) of diluent into the vial of reagent. Do not pour the contents of the diluent vial into the vial of RecombiPlasTin. Replace the stopper and swirl gently. Let sit for 15–20 min at 15–25 °C and invert to mix before use. 3. Factor-deficient plasma: Dissolve the contents of each vial with 1.0 mL of CLSI Type CLRW water or equivalent. Replace the stopper and swirl gently. Ensure complete reconstitution of the product. Keep at 15–25 °C for 30 min, and invert to mix before use. Do not shake. Avoid foam formation. 4. Calibration plasma (only needed if calibrating): Dissolve the contents of each required vial with 1 mL of CLSI CLRW water or equivalent. Replace the stopper and swirl gently. Ensure complete reconstitution of the product. Keep at 15–25 °C for 30 min and invert to mix before use. Do not shake. Avoid foam formation.

3.2 Calibration 1. The calibration procedure should be performed: –– Every 6 months –– With each change of lot number of reagents –– With a major component change –– When indicated by a change in quality control results 272 Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors

2. When calibration is complete: –– Print the curve. –– Record results of controls run post calibration on the curve sheet. 3. File printouts in the appropriate section of the calibration log book.

3.3 Stability 1. RecombiPlasTin 2G: Stability after reconstitution – 10 days at 2–8 °C, 5 days at 15–25 °C in the original vial, or 10 days at 15 °C on the analyzer. 2. Factor-deficient plasma: Stability after reconstitution – 24 h at 2–8 °C in the original vial or 24 h at 15 °C on the analyzer. 3. Calibration plasma: Reconstituted plasma is stable 8 h at 2–8 °C in the original vial on the analyzer. For optimal stability remove reagents from the system and store them at 2–8 °C in the original vial.

3.4 Quality Control 1. Unopened the control is stable until the expiration date shown on the vial when stored at 2–8 °C. Reconstitute controls with the volume of water stated on vial. Swirl and allow to incubate at 15–30 °C for 30 min. Stability after reconstitution: 8 h at 15–25 °C or 24 h at −20 °C in the original vial. Frozen controls may be thawed at 37 °C and gently mixed before use. Do not refreeze. 2. Controls should be tested at least once every shift for any assays run for patient testing during that interval. 3. Controls should also be tested upon reagent changes and after each new calibration curve. 4. Controls should be run in the same manner as the test samples. 5. Corrective action when tolerance limits are exceeded: 6. Recalibration may be necessary if control values are outside the target range. 7. Verify reagent performance. 8. Check instrument performance. 9. Document actions taken to identify and correct the problem before reporting. 10. Indication of deterioration: Normal plasma or controls will show deviations in results from the established laboratory range. Discard and prepare fresh if indicated [6]. Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors 273

3.5 Procedure 1. Factor assays are run with parallelism enabled on ACL®Top 500 CTS Coagulation Analyzer. The ACL Top hemostasis system is a fully automated, bench-top, random-access analyzer, designed specifically for in vitro coagulation and fibrinolysis diagnostic testing in the assessment of thrombosis and hemostasis. It provides results for both direct hemostasis measure- ments and calculated parameters. It has the ability to perform coagulometric (turbidimetric) tests, chromogenic (absor- bance) tests, and immunological tests. For this assay, it uses the coagulometric test. The principle of coagulometric clot detec- tion is used to measure and record the amount of time required for a plasma specimen to clot. This technique assesses coagulation endpoints by measuring change in optical density. 2. Load all required materials and reagents onto analyzer. 3. Load patient sample. 4. Order the test, using the factor parallelism tab. 5. All patient samples are assayed using three dilutions to check for parallelism (inhibitors). The system automatically orders three dilutions. Results for these tests are automatically cor- rected for dilution by the instrument. In non-inhibitor situa- tions these results should correlate within 15%. 6. Any additional dilutions deemed necessary to investigate pos- sible inhibitor presence are performed manually using factor diluent as the diluent and run using the appropriate factor assay. Correction of results for these dilutions would be done manually. 7. Summary. 8. Load reagents. 9. Run controls; if in range proceed with patient testing with parallelism enabled. 10. If controls are out, repeat. If still out, perform calibration.

3.6 Calculations All calculations are automatically performed by the ACL®Top 500 CTS Coagulation Analyzer except in the case of additional manual dilution. Reportable Range: The reportable range in use is <1.0–300% of normal. Values above 300% will be checked by dilution (and result multiplied by dilution factor) but reported as >300%.

3.7 Factor Factor assays on the system are processed using multiple dilutions. Parallelism The dilutions are automatically prepared by the system, and the results are checked for integrity and plotted against the calibration curve. If a sample is void of factor inhibitors, the sample dilution curve and the calibration curve will parallel each other. If a sample contains an inhibitor, the two curves often intersect one another. 274 Serine Protease Assays: Measuring the Enzyme Targets for Serpins, Serine Protease Inhibitors

The ACL®Top 500 CTS Coagulation Analyzer utilizes algorithmic data checks to flag potential samples with potential inhibitors. Manual dilutions are prepared using factor diluent as the diluent. Typically, dilutions of 1:4, 1:8, or 1:16 are used. Run these dilutions with parallelism enabled; results are multiplied by the dilution factor used. Often with an inhibitor, the factor being mea- sured increases with increasing dilutions as the inhibitor is diluted out. The highest factor activity apparent with dilution is the result reported.

References

1. Renné T (2013) The factor XII-driven plasma thromboembolic disease? J Thromb Haemost contact system. In: Marder VJ, Aird WC, 5:1106–1112 Bennett JS, Schulman S, White GC 2nd (eds) 4. Renné T, Gailani D (2007) The role of factor Hemostasis and thrombosis: basic principles XII in hemostasis and thrombosis: clinical and clinical practice, 6th edn. Lippincott implications. Expert Rev Cardiovasc Ther Williams & Wilkins, Philadelphia 5:733–741 2. Gailani D, Broze G (2001) Factor XI and the 5. Biggs R, Rizza C (1984) Human blood coagu- contact system. In: Scriver C, Beaudet A, Sly W, lation, haemostasis and thrombosis, 3rd edn. Valle D, Childs B, Kinzler K, Vogelstein B (eds) Blackwell Scientific Publications, Oxford Metabolic and molecular basis of inherited dis- 6. Zucker S, Cathey MH, West B (1970) ease. McGraw-Hill, New York Preparation of quality control specimens for 3. Gailani D, Renné T (2007) The intrinsic coagulation. Am J Clin Pathol 53:924–927 pathway of coagulation: a target for treating Index

A Immunomodulatory drugs ��73 Inflammation ��2, 73, 74, 134, 143, 144, Alpha-1-antitrypsin (AAT)�� 66–69, 88, 89, 92, 147, 153, 157–159, 162, 164, 166, 167, 173–177, 184, 93, 95, 97, 98, 100–103, 110, 112–115, 119, 126, 127, 197, 198, 201, 208, 224, 225, 233, 237, 238, 256, 263 134, 143–154, 183–194 Inhibition �� 3, 4, 32, 41, 42, 56, 62, 66, Anti-inflammatory treatment �� 147, 164, 173, 184, 224 69, 70, 82, 138–139, 160, 183, 199, 224, 233 Anti-thrombin III (ATIII) �� 2, 4, 66, 67, 69, 70, 135, 158, 256, 268–270 K Antitrypsin �� 10, 11, 22, 25, 34, 43, 66–69, Kinetic �� 6, 56, 62, 65–71, 87, 200 88, 89, 110, 126, 127, 134, 143–154, 183–194, 256 Knock out virus ��74, 75, 78, 80, 83 Autoimmunity �� 146, 150 B L Library preparation ��213–221 Bioinformatics ��43, 59, 213, 214 C M

Cell culture �� 15, 16, 80, 84, 89, 110, 115, 118, Microbiome ��213–221 120, 186, 241 Monoclonal ��90 Cell lines �� 12, 75, 76, 78, 79, 81, 109, 110, Monoclonal antibody ��97, 120, 126, 178 112, 114, 115, 118, 120, 259 Mouse model ��134, 140, 146, 147, 149–151, Cell transfection �� 110, 111, 115, 117, 188 160, 162, 164, 167, 173, 198–201, 207 Clinical trials for serpins ��180, 232, 233, 257, 261, 263 Myxoma virus (MYXV) �� 11, 13, 74–81, 83, Cryo-electron microscopy (cryo-EM) ��10, 26, 27, 84, 225, 238, 244 29, 30, 102 N Cysteine protease ��4, 74 D Negative-stain electron microscopy �� 92–93, 102–105 Neuroinflammation ��223 Deep vein thrombosis (DVT) ��197–200, 205, 207 Neuroprotection ��223–233 Dexamethasone �� 224, 225, 231 Neuroserpin �� 2, 4, 35, 89, 110, Dot blots��127 117, 174, 180 Drug development ��257–259 Neutron crystallography �� 10, 26–29 Next generation sequencing (NGS) ��55, 56, 61, E 213–221 Non-denaturing (native) polyacrylamide gel electrophoresis Enzyme-linked immunosorbent assay (PAGE) �� 124, 125 (ELISA) ��62, 91, 96–98, 110, 117, 120, 148, 150, 185, 186, 188, 189, 191, 207, 233, 260 O F Oligomerization �� 88, 93–95, 98 Förster resonance energy transfer (FRET) �� 89, 92, P 98–101 I Peptide �� 4, 6, 9, 42–44, 57–59, 88, 133–141, 158, 173, 174, 226, 238, 240, 241, 244, 246, 252, 253, Immune �� 2, 4–6, 73, 75, 134, 140, 147, 149, 256, 258–260, 263 153, 157–159, 161, 162, 178, 184, 191, 194, 197, 198, Phage display ��6 202, 224, 238, 257, 263 Phospholipids ��123–132

Plasminogen activator inhibitor-1 (PAI-1) ��2, 4, 43, Small-and wide-angle solution scattering 101, 135, 136, 140, 160, 198, 199, 257 (S/WAS) �� 10, 30, 31 Plasminogen activator inhibitor-2 (PAI-2) �� 198, 199 Solid phase phospholipid binding assays ��124 Plasminogen activators ��2, 4, 11, 12, 43, 44, 101, Spinal cord injury (SCI) ��223–229, 232, 233 134, 135, 160, 197, 198, 256 16S rRNA �� 214, 215 Polyethylenimine (PEI) ��110–112, 114–115, Subdural infusion ��225, 228, 229, 232 118, 119, 240, 251 Systemic lupus erythematosus (SLE) ��149 Polymerase chain reaction (PCR) ��44, 46, 47, T 53–55, 57, 59, 60, 76, 78–80, 100, 164, 165, 175–179, 203, 207, 214–221 Thrombin �� 4, 34, 44, 50, 54, 58–61, 66, 67, Polymerization�� 88, 100, 101, 128, 137 69, 70, 74, 82, 134, 136, 160, 268–270 Post-thrombotic syndrome (PTS) �� 198, 200 Thrombolysis ��2 Poxvirus �� 73, 74, 78 Thrombosis �� 2, 73, 157, 158, 197–209, 224, 267, 273 Protein aggregation �� 12, 256 Transplant��2, 134, 140, 144, 158–173, Proteinases ��12, 41–62, 74, 82, 134, 144 175, 176, 180, 186, 191–193, 256, 260 Protein C inhibitor (PCI) ��42, 123, 124, 126–130 Trypsin �� 3, 66–69, 71, 74, 112, 114, 144, R 185, 190, 191, 251 Type 1 diabetes (T1D) ��146–147, 153, 184, 190 Reactive center loop (RCL) �� 2, 3, 6, 10, 11, U 22, 26, 31, 33, 42–44, 49, 52, 53, 55, 56, 59–61, 66, 73, 77, 88, 133–141, 158, 174, 256 Urokinase-type plasminogen activator (uPA) �� 4, 74, Real-time �� 21, 27, 65–71, 92, 99, 100, 203, 207 82, 134, 136, 160, 197–199, 257 Rheumatoid arthritis (RA) �� 146–149, 184 V S Vasculitis �� 139, 158, 162, 164, 173 Selectivity ��43, 62 Vena cava �� 166, 168, 199–202, 205–209 Serine protease inhibitors ��2, 65, 73, 74, Venous thrombus resolution �� 197–199, 201–209 123, 133, 157, 180, 198, 267–274 Virus ��5, 6, 11, 13, 30, 35, 42, 73–84, 136, Serine proteases ��2, 4, 65, 66, 73, 74, 123, 158, 160, 162, 164, 173, 180, 183–194, 225, 237–253, 133, 136, 157, 160, 165, 180, 197, 256, 267–274 257, 263 Serp-1 ��74, 75, 81, 82, 134, 135, 139, 140, 160, 162, 171, 174, 180, 225, 231, 232, 257, 260, 262, 263 W Serpin conformations ��28, 33, 34, 120 Western blot ��77, 82, 89–91, 95–96, 110, Serpinopathy ��2, 5, 6, 27, 30, 87, 109–118, 120, 134, 256 118, 120, 124, 125, 128–130, 244, 251 Serpin polymers ��87–96, 98–100, 102, 104–106, 118, 120 Serpins ��2, 9, 41, 66, 73, 87, 109, 123, 133, X 144, 157, 183, 198, 214, 224, 237–253, 256, 257, 268 Serpin structures ��2, 4, 9, 27, 35, 42, 77, 158 X-ray crystallography��9–35