and Clotting are in Balance

Disorders of Dr. Raymond L. Lobins Cox health – Springfield, Missouri

Vasoconstriction

! 1. Vasoconstriction of a damaged blood vessel slows the flow of blood and thus helps to limit blood loss. This process is mediated by: ! Local controls. Vasoconstrictors such as thromboxane are released at the site of the injury. ! Systemic control. Epinephrine released by the adrenal glands stimulates general vasoconstriction. pathways Adhesion 1.Cross links to (PLT)

! vWF released from and floating in blood binds with collagen. ! Rolling PLT Bind to vWF via gpIb/V/ IX receptor. ! PLT bound to vWF changes shape, releases granules (alpha and dense). ! PLT shape change also opens the gp IIb/IIIa receptor to allow binding with .

Platelet activation - Platelet Granules Platelet Aggregation

! Alpha Granules – there are many ! Release of granules supply many of types of alpha granules that the products needed to attract contain varied products. But more platelets, maintain vaso- alpha granules do contain vWF, FV, constriction, and start secondary and FXIII and fibrinogen! hemostasis.

! Dense granules - contain a range of small molecules such as ADP, ! ADP – activates cell via P2Y12 ATP, GDP, serotonin, receptor, involved in Ca++ pyrophosphate, magnesium and regulation. calcium ! Platelets also secrete Thromboxane A2 – blocks prostacyclin (via gpVI). vasoconstriction and activation of IIb/IIIa receptor. ! Serotonin – vasoconstriction. Signs and Symptoms of defects in primary hemostasis

Disorders of Hemostasis Primary Hemostasis

Primary hemostasis disorders Vascular Anomalies

! Structural anomalies - such as ! hereditary hemorrhagic telangiectasia. ! Vascular anomalies. ! disorders of the connective tissue (including Ehlers–Danlos disease and osteogenesis imperfecta). – these are your “rubber men”

! Thrombocytopenia. ! small vessel vasculitis

! . ! Generalized vascular fragility dominates the clinical picture. ! Can lead to severe varicosities and arterial rupture, which may cause sudden death (usually in the third or fourth decade of life). ! Platelet function disorders. Thrombocytopenia Von Willebrand disease

! Most common coagulopathy (1 -2% of population). ! Von Willebrand is stored in endothelial cells and platelets. ! Composed of repeating multimers. ! Most types are Autosomal dominant (types 2N and 3 are recessive). ! Normal plasma corrects the abnormal bleeding time. ! Ristocetin co-factor measures activity of vWF.

Alterations in vWF Concentration Types of vWD

! INCREASED ! DECREASD

! Pregnancy ! Wilms tumor ! Physical Stress ! Type O ! Epinephrine ! VSD ! Emotional Stress ! Congenital Heart dz. ! Estrogens ! Monoclonal ! Thyroid hormones gammopathies ! Exercise ! Myelo/lympho- ! Elderly proliferative disorders ! Diabetes Von Willebrand disease Von Willebrand disease screening tests

! 182kb produce a 2813 amino acid ! Location – 12 at single peptide pro-VWF 12p13.2 ! Bleeding time – insensitive & nonspecific ! vWF is composed of repeating ! Pseudo-vWF location - chromosome multimers of dimers 500kD-10,00kD. 22 ! Requires cleaving for active vWF. ! Platelet function analyzer-100 (PFA-100) 1996 2 types of cartridges (col/epi & col/adp). ! Cleavage by ADAMTS13 @ A2 ! Secreted in 2 ways inactivate vWF (regulated & unregulated). Blood passed thru cartridge at high pressures – ! Smaller multimers and subunits can ! Platelets secretion is only via time to clot is measured. bind to factor VIII at domains C & D regulated pathway…..therefore….. Closure time is increased in primary hemostasis disorders. ! Factor VIII/vWF complex protects ! All circulating vWF is by endothelial For type1 vWD col/epi – more sensitive & specific. Factor VIII from and excretion. extends it’s half life. ! ABO typing

! PTT – prolonged if VIII< 30% of normal

Von Willebrand disease specific tests Von Willebrand disease type specific tests

!vWF antigen – measures quantity. !Ristocetin induced platelet aggregation (RIPA)- To Some labs have separate references for type O. determine qualitative defect in vWF binding !Ristocetin cofactor – measures activity of vWF Looking for spontaneous binding or binding at low ristocetin cleaves vWF allowing it to bind to gpIb. concentration (IIB or pseudo-vWD) !Collagen binding assay –measures how well plasma vWF binds collagen. !vWF multimers- detected by gel electrophoresis. !Factor VIII level Can detect absence of lg multimers (IIa) or if larger than normal (Vicenza, TTP, neonates) Algorhythm Platelet function disorders - Storage pool deficiency (SPD).

! Rare. ! Deficiencies in platelet secretion granules, changing the contents of ! dense granules (δ-SPD) = (ADP and Serotonin), alpha-granules (α-SPD or gray platelet syndrome), or both (αδ-SPD). ! Can be idiopathic or part of a complex disorder such as Hermansky–Pudlak syndrome, Chediak-Higashi syndrome, Wiskott-Aldrich syndrome, and thrombocytopenia-absent radius (TAR).

! Bleeding tendency is usually mild.

Platelet function disorders Platelet function disorders

! Glanzmann thrombasthenia ! Bernard–Soulier syndrome ! Rare. ! Rare. Autosomal recessive. Autosomal recessive. ! Defect in the platelet membrane receptor GPIIb-IIIa ! Defect in one of the components of the GP Ib-IX-V complex. (the main fibrinogen receptor on the platelet surface). Abnormally rapid removal of the bizarre platelets may be responsible for Ineffective platelet aggregation. thrombocytopenia

! Genetic predisposition (French Gypsies, Iraqi Jews, Jordanian Arabs, and South Indians). ! Noted by high phospholipid content in platelets. ! Normal platelet morphology and normal platelet count. ! Giant platelets & thrombocytopenia. ! Do not aggregate to ristocetin. ! Do not aggregate to ristocetin. Secondary Coagulation – Plug Stability

Disorders of Hemostasis Secondary Hemostasis

Secondary Coagulation Secondary Coagulation ! Extrinsic pathway: ! Intrinsic pathway: ! (cofactor) and FVII (pro- ! Enzymatic coagulation factors: enzyme; FVIIa is the enzyme) + (Ca2+) ! FXII, FXI, and FIX and a cofactor ! Common pathway: (non-enzyme): FVIII, along with ! The TF-FVIIa complex activates FX Ca2+ . ! Enzymatic coagulation factors: FX and prothrombin (FII) with FV (a cofactor) and also activate FIX & Ca2+ ! Occurs on the phospholipid ! Under physiologic conditions (blood surface(PS). ! Works on a Phospholipid surface (PS) and results in fibrinogen to . vessel injury), FXa is generated by the TF-FVIIa complex on the surface ! The ultimate product of the ! FXa, with the aid of its cofactor FVa, activates prothrombin to . of fibroblasts. intrinsic pathway is activated FIX ! Thrombin cleaves fibrinogen to soluble fibrin, which is then crosslinked to ! (Factor Ixa). Initiation of thrombin generation insoluble fibrin by FXIIIa (FXIII is also activated by thrombin). occurs through extrinsic pathway, ! Thus both intrinsic & extrinsic which is expressed on fibroblasts. pathways converge at the ! This generates small amounts of activation of FX. thrombin and Factor IXa – leads to ! however the site of activation of thrombin burst. FX differs (fibroblast for the extrinsic pathway and platelet for the intrinsic pathway). Defects in Secondary Hemostasis

Defects in Secondary Hemostasis (Disorders of Fibrin Formation)

Disorders of Diagnostic testing – in General of Fibrin Formation ! Isolated High PTT – think deficiency in factors VIII, IX, and / or XI.

! Hereditary vs acquired ! Isolated High PT – think deficiency in factor VII. ! Quantitative vs qualitative deficiencies ! Laboratory screening tests (PT, APTT) ! Both PT and PTT high – think deficiency in factors II, V, or X. ! Does not differentiate quantitative vs qualitative disorders ! Qualitative abnormal proteins will ! Prolong clotting test ! Both PT and PTT high and thrombin time increased – think fibrinogen deficiency. ! Being recognized by immunologically-based procedures ! Activity assays ! ! Essential when screening for deficiencies PT, PTT, thrombin time, and bleeding time all normal – think factor XIII. Hemophilia Hemophilia

○ Hemophilia A ● Factor VIII Deficiency - 1/5000 ○ Antihemophilic Factor ! Insufficient generation of thrombin ○ X-linked recessive disorder ! Due to lack of FIXa/VIIIa complex through the intrinsic pathway. ○ Most common type of hemophilia ! Bleeding severity complicated by excessive ○ Hemophilia B ● Factor IX Deficiency – 1/20,000 ○ Christmas Factor (from family of first patients diagnosed with the disorder) ! Clinical severity corresponds with level of factor activity ○ X-linked recessive disorder ○ Hemophilia C ● Factor XI Deficiency – 1/100,000 ● Autosomal recessive disorder seen primarily in the Ashkenazi Jewish population ● Symptoms range from mild to severe

Hemophilia severities Hemophilia – Treatment

! Severe hemophilia ! Replacement of clotting factor to achieve hemostasis ! Annual cost for patient with severe hemophilia ! Factor coagulant activity <1% of normal ! $20,000-100,000 ! Frequent spontaneous bleeding into joints and soft tissues ! Various products available ! Prolonged bleeding with trauma or surgery ! Plasma-derived low, intermediate and high purity products ! Plasma-derived ultrapure products ! Moderate hemophilia ! Ultrapure recombinant products ! Factor coagulant activity 1-5% of normal ! Replacement products – benefits vs risks ! Occasional spontaneous bleeding ! Blood-born pathogens ! Excessive bleeding with surgery or trauma ! Hepatitis A, B, C, G; HIV, Parvovirus B-19 ! Thrombotic complications with some F-IX concentrations ! Mild hemophilia ! Development of alloantibody inhibitors ! Factor coagulant activity >5% of normal ! Neutralize coagulant effects of replacement therapy ! Usually no spontaneous bleeding ! Excessive bleeding with surgery or trauma Factor Replacement ! Plasma derived (purified) – Alphanate, Koate, Wilate, Humate-P. T1/2 = 12hr, thus treatment = 3x/week.

! Recombinant VIII – Made from DNA in animal cell lines. – (more immunogenic). Recombinate, Kogenate, Helixate, Advate. T1/2 = 12hr, thus treatment = 3x/week.

! Recombinant VIII Fc fusion – Eloctate. T1/2 = 18hr, thus treatment = q4-5 days.

! Recombinant IX - Benefix (T1/2 = 33hr), thus treatment = 2x/week.

! Recombinant IX Fc fusion protein or IX-FP (albumin). Alprolix (T1/2 = 82hr) or Idelvion (T1/2 = 102hr).

Treatment options - historically Replacement dosing ! Historically Prothrombin complex concentrates (PCC) 3F – 4F. (FFP is not a concentrate).

! Dose calculations – ! Enriched concentrates of factors II, IX, X, (VIIa – 4F, some with and s). ! 30-40% for most mild hemorrhages. ! Named by there level of factor IX in them. > 50% for severe bleeds (trauma / major dental surgery / major surgery). ! Are not primarily used for hemophilia treatment any longer. 80-100% in life-threatening hemorrhage. Usually used for bleeding or anticoagulation reversal. ! Typically one dose (rarely may need more than one dose). ! Hospitalization is reserved for severe or life-threatening bleeds, such as large- soft tissue bleeds; retroperitoneal hemorrhage or other internal bleeding; and hemorrhage related to head injury, surgery, or dental work. ! Primary benefit over FFP = volume infused and time to correction (30minutes vs. up to 24hr).

! Of note: 4F-PCC did reverse “lab effects” in healthy donors from Rivaroxiban. Indication or Site of Bleeding Factor level Desired, % FVIII Dose, IU/kg* Comment

Severe epistaxis; mouth, lip, tongue, or dental work 20-50 10-25 Consider (Amicar), 1-2 d Dosing principles – standard products Joint (hip or groin) 40 20 Repeat transfusion in 24-48 h

! Factor VIII – 1 iu/kg = 2% increase. ! Factor IX – 1iu/kg = 1% increase (plasma) Soft tissue or muscle 20-40 10-20 No therapy if site small and not enlarging (transfuse if enlarging) 0.75% (recombinant) Muscle (calf and forearm) 30-40 15-20 None

Muscle deep (thigh, hip, iliopsoas) 40-60 20-30 Transfuse, repeat at 24 h, then as needed

! Joint bleed – 50iu/kg (repeat prn) Neck or throat 50-80 25-40 None ! Joint bleed – 25iu/kg (repeat prn) Hematuria 40 20 Transfuse to 40% then rest and hydration

! Life threat – 100iu/kg (repeat prn) Laceration 40 20 Transfuse until wound healed ! Life threat – 50iu/kg (repeat prn) GI or retroperitoneal bleeding 60-80 30-40 None

! Surgery – 100iu/kg x 1 Head trauma (no evidence of CNS bleeding) 50 25 None ! Surgery – 50iu/kg x 1

Head trauma (probable or definite CNS bleeding, eg, headache, Maintain peak and trough factor levels at 100% and 50% for 14 d if 100 50 † ! vomiting, neurologic signs) CNS bleeding documented ! Prophylaxis – 25iu/kg qod Prophylaxis – 50iu/kg 2x/wk.

Trauma with bleeding, surgery† 80-100 50 10-14 d

Acquired Factor VIII antibodies Acquired Factor IX antibodies

! Occurs in about 30% of Hemophillia A patients. ! Occurs less often vs. Factor VIII. ! Severity measured in Bethesda units. ! Severity measured in Bethesda units. ! Occur more often with use of recombinant products than plasma based ! Occur more often with use of recombinant products than plasma based products (45% vs 27% - NEJM 5/26/16). products. ! Treatment options: ! Treatment options: the same much only about ½ as effective. 1. Bypassing agents (i.e. Factor VIIa or prothrombin complex concentrates). 1. Bypassing agents (i.e. Factor VIIa or prothrombin complex concentrates). 2. Induce immune tolerance ( works about 70% of the time). 2. Induce immune tolerance 3. (bispecific antibody which mimics cofactor functionof FVIII)? 3. Emicizumab (bispecific antibody which mimics cofactor functionof FVIII)?

! Also holds true for Acquired Hemophillia A (1/1.3 million) ! Rituximab can also be tried if needed. ! Acquired antibodies to FVIII in patients without Hemophillia A at birth. ! Risks – increased age, pregnancy, cancer, auto-immune diseases. Coagulation screening tests in congenital deficiencies Platlet PT APTT PFA TT Congenital Deficiency count

N N N N N XIII, mild deficiency of any factor, plasminogen activator inhibitor-1, α2 anti- N A N N N VII

N N A N N XII, XI, IX, VIII, , high molecular weight

N A A N N X, V, II

N A A N A Fibrinogen

N N A or N A or N N Von Willibrands

Factor XIII deficiency Acquired coagulopathy

! Factor XIII consists of two non-identical-polypeptide subunits, the "a" chain and the "b“. ! ! The activated form of factor XIII catalyzes the formation of covalent bonds between the Numerous acquired conditions and gamma chains and alpha chains of fibrin (cross-linking). ! Cross-linking by Factor XIII does not affect PT and APTT. ! disease, Alcoholism, or even simple aging. ! homozygous-recessive patients lack the “a” chains. ! Numerous medications can affect coagulopathy ! Ecchymoses, hematomas, hemarthroses, and prolonged bleeding after superficial ! Acquired conditions – DIC, TTP, Sepsis, ITP, Cancers, etc. wounds, dental extractions or surgery. ! Tocilizumab Induced Acquired Factor XIII Deficiency. ! See a Hematologist and/or correct the underlining cause. ! Of note: there are 2 factor XIII products on the market. Thank You Questions?